Characterization of D-tagatose-3-epimerase from Rhodobacter sphaeroides that converts D-fructose into D-psicose.

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Zhang, Longtao; Mu, Wanmeng; Jiang, Bo; Zhang, Tao

2009-06-01

A non-characterized gene, previously proposed as the D-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with D-fructose and decreased for other substrates in the order: D-tagatose, D-psicose, D-ribulose, D-xylulose and D-sorbose. Its activity was maximal at pH 9 and 40 degrees C while being enhanced by Mn(2+). At pH 9 and 40 degrees C, 118 g D-psicose l(-1) was produced from 700 g D-fructose l(-1) after 3 h.

Role of the Irr protein in the regulation of iron metabolism in Rhodobacter sphaeroides.

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Verena Peuser

Full Text Available In Rhizobia the Irr protein is an important regulator for iron-dependent gene expression. We studied the role of the Irr homolog RSP_3179 in the photosynthetic alpha-proteobacterium Rhodobacter sphaeroides. While Irr had little effect on growth under iron-limiting or non-limiting conditions its deletion resulted in increased resistance to hydrogen peroxide and singlet oxygen. This correlates with an elevated expression of katE for catalase in the Irr mutant compared to the wild type under non-stress conditions. Transcriptome studies revealed that Irr affects the expression of genes for iron metabolism, but also has some influence on genes involved in stress response, citric acid cycle, oxidative phosphorylation, transport, and photosynthesis. Most genes showed higher expression levels in the wild type than in the mutant under normal growth conditions indicating an activator function of Irr. Irr was however not required to activate genes of the iron metabolism in response to iron limitation, which showed even stronger induction in the absence of Irr. This was also true for genes mbfA and ccpA, which were verified as direct targets for Irr. Our results suggest that in R. sphaeroides Irr diminishes the strong induction of genes for iron metabolism under iron starvation.

Ammonium and methylammonium transport in Rhodobacter sphaeroides

International Nuclear Information System (INIS)

Cordts, M.L.; Gibson, J.

1987-01-01

Rhodobacter spheroides maintained intracellular ammonium pools of 1.1 to 2.6 mM during growth in several fixed nitrogen sources as well as during diazotrophic growth. Addition of 0.15 mM NH 4 + to washed, nitrogen-free cell suspensions was followed by linear uptake of NH 4 + from the medium and transient formation of intracellular pools of 0.9 to 1.5 mM NH 4 + . Transport of NH 4 + was shown to be independent of assimilation by glutamine synthetase because intracellular pools of over 1 mM represented NH 4 + concentration gradients of at least 100-fold across the cytoplasmic membrane. Ammonium pools of over 1 mM were also found in non-growing cell suspensions in nitrogen-free medium after glutamine synthetase was inhibited with methionine sulfoximine. In NH 4 + -free cell suspensions, methylammonium ( 14 CH 3 NH 3 + ) was taken up rapidly, and intracellular concentrations of 0.4 to 0.5 mM were maintained. The 14 CN 3 NH 3 + pool was not affected by methionine sulfoximine. Unlike NH 4 + uptake, 14 CH 3 NH 3 + uptake in nitrogen-free cell suspensions was repressed by growth in NH 4 + . These results suggest that R. sphaeroides may produce an NH 4 + -specific transport system in addition to the NH 4 + / 14 CH 3 NH 3 + transporter. This second transporter is able to produce normal-size NH 4 + pools but has very little affinity for 14 CH 3 NH 3 + and is not repressed by growth in high concentrations of NH 4 +

Transient grating spectroscopy in photosynthetic purple bacteria Rhodobacter sphaeroides 2.4.1

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Sugisaki, Mitsuru, E-mail: mitsuru@sci.osaka-cu.ac.j [CREST-JST and Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585 (Japan); Fujiwara, Masazumi; Fujii, Ritsuko [CREST-JST and Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585 (Japan); Nakagawa, Katsunori; Nango, Mamoru [CREST-JST and Graduate School of Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Cogdell, Richard J. [Glasgow Biomedical Research Centre, IBLS, University of Glasgow, 126 University Place, Glasgow G12 8TA, Scotland (United Kingdom); Hashimoto, Hideki [CREST-JST and Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585 (Japan)

2009-12-15

The vibronic coherence of photosynthetic pigment-protein complexes has been investigated by means of transient grating spectroscopy using sub 20 fs optical pulses. In the present work, we focus our attention on the LH2 antenna complexes from Rhodobacter sphaeroides 2.4.1 because the information about their structure investigated by the electron and atomic force microscopy is available and the electric levels of pigments are well resolved, resulting in clear absorption spectrum. The vibronic coherent oscillations with a period of a few tens of femtoseconds have been clearly observed. We found that the temporal change of the coherent oscillations reflects the vibrational relaxation in the ground state. Calculations based on the Brownian oscillator model were performed under the impulsive excitation limit. The spectral density has been determined from the Raman measurement of spheroidene. Good agreement between the calculation and the experimental results has been achieved in the linear absorption spectrum and transient grating signal, which strongly supports the validity of our model.

Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

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Verméglio, André; Lavergne, Jérôme; Rappaport, Fabrice

2016-01-01

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

Correlation of paramagnetic states and molecular structure in bacterial photosynthetic reaction centers: The symmetry of the primary electron donor in Rhodopseudomonas viridis and Rhodobacter sphaeroides R-26

International Nuclear Information System (INIS)

Norris, J.R.; Budil, D.E.; Gast, P.; Chang, C.H.; El-Kabbani, O.; Schiffer, M.

1989-01-01

The orientation of the principal axes of the primary electron donor triplet state measured in single crystals of photosynthetic reaction centers is compared to the x-ray structures of the bacteria Rhodobacter (Rb.) sphaeroides R-26 and Rhodopseudomonas (Rps.) viridis. The primary donor of Rps. viridis is significantly different from that of Rb. sphaeroides. The measured directions of the axes indicate that triplet excitation is almost completely localized on the L-subunit half of the dimer in Rps. viridis but is more symmetrically distributed on the dimeric donor in Rb. sphaeroides R-26. The large reduction of the zero field splitting parameters relative to monomeric bacteriochlorophyll triplet in vitro suggests significant participation of asymmetrical charge transfer electronic configurations in the special pair triplet state of both organisms

Timescales of Coherent Dynamics in the Light Harvesting Complex 2 (LH2) of Rhodobacter sphaeroides.

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Fidler, Andrew F; Singh, Ved P; Long, Phillip D; Dahlberg, Peter D; Engel, Gregory S

2013-05-02

The initial dynamics of energy transfer in the light harvesting complex 2 from Rhodobacter sphaeroides were investigated with polarization controlled two-dimensional spectroscopy. This method allows only the coherent electronic motions to be observed revealing the timescale of dephasing among the excited states. We observe persistent coherence among all states and assign ensemble dephasing rates for the various coherences. A simple model is utilized to connect the spectroscopic transitions to the molecular structure, allowing us to distinguish coherences between the two rings of chromophores and coherences within the rings. We also compare dephasing rates between excited states to dephasing rates between the ground and excited states, revealing that the coherences between excited states dephase on a slower timescale than coherences between the ground and excited states.

Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

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Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston; Niedzwiedzki, Dariusz M.; Zhang, Hao; Qian, Pu; Vasilev, Cvetelin; Grayson, Katie J.; Jackson, Philip J.; Martin, Elizabeth C.; Li, Ying; Holten, Dewey; Neil Hunter, C.

2015-01-01

Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon–carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N = 10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2′-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC–LH1–PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2′-diketo-spirilloxanthin (15 conjugated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C bonds; N = 15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N = 9; 94%), spheroidene (N = 10; 96%) and spheroidenone (N = 11; 95%), whereas intermediate values were measured for lycopene (N = 11; 64%), rhodopin (N = 11; 62%) and spirilloxanthin (N = 13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the

Identification of the upper exciton component of the B850 bacteriochlorophylls of the LH2 antenna complex, using a B800-free mutant of Rhodobacter sphaeroides.

NARCIS (Netherlands)

Koolhaas, M.H.C.; Frese, R.N.; Fowler, G.J.S.; Bibby, T.S.; Georgakopoulou, S.; van der Zwan, G.; Hunter, C.N.; van Grondelle, R.

1998-01-01

In this paper, we report the circular dichroism (CD) spectra of two types of LH2 -only mutants of Rhodobacter sphaeroides. In the first, only the wild type LH2 is present, while in the second, the B800 binding site of LH2 has been either destabilized or removed. For the first time, we have

Engineering of a calcium-ion binding site into the RC-LH1-PufX complex of Rhodobacter sphaeroides to enable ion-dependent spectral red-shifting.

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Swainsbury, David J K; Martin, Elizabeth C; Vasilev, Cvetelin; Parkes-Loach, Pamela S; Loach, Paul A; Neil Hunter, C

2017-11-01

The reaction centre-light harvesting 1 (RC-LH1) complex of Thermochromatium (Tch.) tepidum has a unique calcium-ion binding site that enhances thermal stability and red-shifts the absorption of LH1 from 880nm to 915nm in the presence of calcium-ions. The LH1 antenna of mesophilic species of phototrophic bacteria such as Rhodobacter (Rba.) sphaeroides does not possess such properties. We have engineered calcium-ion binding into the LH1 antenna of Rba. sphaeroides by progressively modifying the native LH1 polypeptides with sequences from Tch. tepidum. We show that acquisition of the C-terminal domains from LH1 α and β of Tch. tepidum is sufficient to activate calcium-ion binding and the extent of red-shifting increases with the proportion of Tch. tepidum sequence incorporated. However, full exchange of the LH1 polypeptides with those of Tch. tepidum results in misassembled core complexes. Isolated α and β polypeptides from our most successful mutant were reconstituted in vitro with BChl a to form an LH1-type complex, which was stabilised 3-fold by calcium-ions. Additionally, carotenoid specificity was changed from spheroidene found in Rba. sphaeroides to spirilloxanthin found in Tch. tepidum, with the latter enhancing in vitro formation of LH1. These data show that the C-terminal LH1 α/β domains of Tch. tepidum behave autonomously, and are able to transmit calcium-ion induced conformational changes to BChls bound to the rest of a foreign antenna complex. Thus, elements of foreign antenna complexes, such as calcium-ion binding and blue/red switching of absorption, can be ported into Rhodobacter sphaeroides using careful design processes. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Electrochemical determination of hydrogen peroxide using Rhodobacter capsulatus cytochrome c peroxidase at a gold electrode

NARCIS (Netherlands)

De Wael, K.; Buschop, H.; Heering, H.A.; De Smet, L.; Van Beeumen, J.; Devreese, B.; Adriaens, A.

2007-01-01

We describe the redox behaviour of horse heart cytochrome c (HHC) and Rhodobacter capsulatus cytochrome c peroxidase (RcCCP) at a gold electrode modified with 4,4?-bipyridyl. RcCCP shows no additional oxidation or reduction peaks compared to the electrochemistry of only HHC, which indicates that it

Ammonium and methylammonium transport in Rhodobacter sphaeroides

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Cordts, M.L.; Gibson, J.

1987-04-01

Rhodobacter spheroides maintained intracellular ammonium pools of 1.1 to 2.6 mM during growth in several fixed nitrogen sources as well as during diazotrophic growth. Addition of 0.15 mM NH/sub 4//sup +/ to washed, nitrogen-free cell suspensions was followed by linear uptake of NH/sub 4//sup +/ from the medium and transient formation of intracellular pools of 0.9 to 1.5 mM NH/sub 4//sup +/. Transport of NH/sub 4//sup +/ was shown to be independent of assimilation by glutamine synthetase because intracellular pools of over 1 mM represented NH/sub 4//sup +/ concentration gradients of at least 100-fold across the cytoplasmic membrane. Ammonium pools of over 1 mM were also found in non-growing cell suspensions in nitrogen-free medium after glutamine synthetase was inhibited with methionine sulfoximine. In NH/sub 4//sup +/-free cell suspensions, methylammonium (/sup 14/CH/sub 3/NH/sub 3//sup +/) was taken up rapidly, and intracellular concentrations of 0.4 to 0.5 mM were maintained. The /sup 14/CN/sub 3/NH/sub 3//sup +/ pool was not affected by methionine sulfoximine. Unlike NH/sub 4//sup +/ uptake, /sup 14/CH/sub 3/NH/sub 3//sup +/ uptake in nitrogen-free cell suspensions was repressed by growth in NH/sub 4//sup +/. These results suggest that R. sphaeroides may produce an NH/sub 4//sup +/-specific transport system in addition to the NH/sub 4//sup +///sup 14/CH/sub 3/NH/sub 3//sup +/ transporter. This second transporter is able to produce normal-size NH/sub 4//sup +/ pools but has very little affinity for /sup 14/CH/sub 3/NH/sub 3//sup +/ and is not repressed by growth in high concentrations of NH/sub 4//sup +/.

A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides.

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Hendrischk, Anne-Kathrin; Frühwirth, Sebastian Walter; Moldt, Julia; Pokorny, Richard; Metz, Sebastian; Kaiser, Gebhard; Jäger, Andreas; Batschauer, Alfred; Klug, Gabriele

2009-11-01

Blue light receptors belonging to the cryptochrome/photolyase family are found in all kingdoms of life. The functions of photolyases in repair of UV-damaged DNA as well as of cryptochromes in the light-dependent regulation of photomorphogenetic processes and in the circadian clock in plants and animals are well analysed. In prokaryotes, the only role of members of this protein family that could be demonstrated is DNA repair. Recently, we identified a gene for a cryptochrome-like protein (CryB) in the alpha-proteobacterium Rhodobacter sphaeroides. The protein lacks the typical C-terminal extension of cryptochromes, and is not related to the Cry DASH family. Here we demonstrate that CryB binds flavin adenine dinucleotide that can be photoreduced by blue light. CryB binds single-stranded DNA with very high affinity (K(d) approximately 10(-8) M) but double-stranded DNA and single-stranded RNA with far lower affinity (K(d) approximately 10(-6) M). Despite of that, no in vitro repair activity for pyrimidine dimers in single-stranded DNA could be detected. However, we show that CryB clearly affects the expression of genes for pigment-binding proteins and consequently the amount of photosynthetic complexes in R. sphaeroides. Thus, for the first time a role of a bacterial cryptochrome in gene regulation together with a biological function is demonstrated.

Role of the PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 2. PufX is required for efficient ubiquinone/ubiquinol exchange between the reaction center QB site and the cytochrome bc1 complex.

Science.gov (United States)

Barz, W P; Verméglio, A; Francia, F; Venturoli, G; Melandri, B A; Oesterhelt, D

1995-11-21

The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides because it is required for multiple-turnover electron transfer under anaerobic conditions [see accompanying article; Barz, W. P., Francia, F., Venturoli, G., Melandri, B. A., Verméglio, A., & Oesterhelt, D. (1995) Biochemistry 34, 15235-15247]. In order to understand the molecular role of PufX, light-induced absorption spectroscopy was performed using a pufX- mutant, a pufX+ strain, and two suppressor mutants. We show that the reaction center (RC) requires PufX for its functionality under different redox conditions than the cytochrome bc1 complex: When the kinetics of flash-induced reduction of cytochrome b561 were monitored in chromatophores, we observed a requirement of PufX for turnover of the cytochrome bc1 complex only at high redox potential (Eh > 140 mV), suggesting a function of PufX in lateral ubiquinol transfer from the RC. In contrast, PufX is required for multiple turnover of the RC only under reducing conditions: When the Q pool was partially oxidized in vivo using oxygen or electron acceptors like dimethyl sulfoxide or trimethylamine N-oxide, the deletion of PufX had no effect on light-driven electron flow through the RC. Flash train experiments under anaerobic in vivo conditions revealed that RC photochemistry does not depend on PufX for the first two flash excitations. Following the third and subsequent flashes, however, efficient charge separation requires PufX, indicating an important role of PufX for fast Q/QH2 exchange at the QB site of the RC. We show that the Q/QH2 exchange rate is reduced approximately 500-fold by the deletion of PufX when the Q pool is nearly completely reduced, demonstrating an essential role of PufX for the access of ubiquinone to the QB site. The fast ubiquinone/ubiquinol exchange is partially restored by suppressor mutations altering the macromolecular antenna structure. These results suggest an indirect role of PufX in

Mathematical model for the analytical signal of an herbicide sensor based on the reaction centre of Rhodobacter sphaeroides.

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Andreu, Yolanda; Baldini, Francesco; Giannetti, Ambra; Mencaglia, Andrea

2005-01-30

This paper introduces a mathematical model which makes it possible both to determine the concentration of photosynthetic herbicides and to obtain a quantitative parameter in order to compare their activity using a previously described sensing system. The working principle involves the changes in absorption properties at 860nm of the reaction centre (RC) isolated from the bacteria Rhodobacter sphaeroides when photosynthetic herbicides are present. The method has been used for the determination and activity comparison of five photosynthetic herbicides: diuron, atrazine, terbutryn, terbuthylazine and simazine. Detection limits obtained were 2.2, 0.75, 0.046, 0.25, and 1.4muM, respectively. The resulting order for the different herbicides according to their action on RC was: terbutryn > terbuthylazine > atrazine > simazine > diuron.

Effect of pH on optimization of photofermentative hydrogen production by co-culture of Rhodobacter sphaeroides-NMBL-02 and Bacillus firmus-NMBL-03.

Science.gov (United States)

Pandey, A; Dolly, S; Semwal, D; Pandey, A

2017-07-31

Rhodobacter sphaeroides NMBL-02, photosynthetic purple non sulfur (PNS) bacteria and associated Bacillus firmus NMBL-03 were isolated from water sample collected from 15-20 inches beneath the surface of ponds from Northern region of India in modified Sistrom's media (120 ml) containing 3 g/L malate and 1.2 g/L ammonium sulfate. The isolation was done in air tight serum bottles (120 ml) under tungsten bulb (1.8 kLux light intensity) at 30 oC ± 2 oC. The PNS and heterotrophic bacteria associated with the culture was purified by clonal selection method and characterized by 16S rDNA sequencing. The PNS isolate was identified as Rhodobacter sphaeroides NMBL-02 (ID: 1467407, Accession BANKIT: JN256030) and associated heterotroph as Bacillus firmus NMBL-03 (Gene Bank Accession no.: JN 256029). The effect of initial medium pH on optimization of hydrogen production was investigated in batch process. The maximum hydrogen potential and hydrogen production rate was 2310 ± 55 ml/L and 4.75 ml/L culture/h respectively using glutamate (1.7 mmol/L) as nitrogen source and malate (22.38 mmol/L) as carbon source with 76.39% malate conversion efficiency at initial medium pH 5.0. This co-culture has the ability to produce significant amount of hydrogen in the pH range of 5.0 to 10.0 with 76.39% to 35.71% malate conversion respectively.

Improved hydrogen production by uptake hydrogenase deficient mutant strain of Rhodobacter sphaeroides O.U.001

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Kars, Goekhan; Guenduez, Ufuk; Yuecel, Meral [Department of Biological Sciences, Middle East Technical University, 06531 Ankara (Turkey); Rakhely, Gabor; Kovacs, Kornel L. [Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged (Hungary); Eroglu, Inci [Department of Chemical Engineering, Middle East Technical University, 06531 Ankara (Turkey)

2008-06-15

Rhodobacter sphaeroides O.U.001 is a purple non-sulfur bacterium producing hydrogen under photoheterotrophic conditions. Hydrogen is produced by Mo-nitrogenase enzyme and substantial amount of H{sub 2} is reoxidized by a membrane-bound uptake hydrogenase in the wild type strain. To improve the hydrogen producing capacity of the cells, a suicide vector containing a gentamicin cassette in the hupSL genes was introduced into R. sphaeroiodes O.U.001 and the uptake hydrogenase genes were destroyed by site directed mutagenesis. The correct integration of the construct was confirmed by uptake hydrogenase activity measurement, PCR and subsequent sequence analysis. The wild type and the mutant cells showed similar growth patterns but the total volume of hydrogen gas evolved by the mutant was 20% higher than that of the wild type strain. This result demonstrated that the hydrogen produced by the nitrogenase was not consumed by uptake hydrogenase leading to higher hydrogen production. (author)

Photodynamics of the small BLUF protein BlrB from Rhodobacter sphaeroides.

Science.gov (United States)

Zirak, P; Penzkofer, A; Schiereis, T; Hegemann, P; Jung, A; Schlichting, I

2006-06-01

The BLUF protein BlrB from the non-sulphur anoxyphototrophic purple bacterium Rhodobacter sphaeroides is characterized by absorption and emission spectroscopy. BlrB expressed from E. coli binding FAD, FMN, and riboflavin (called BrlB(I)) and recombinant BlrB containing only FAD (called BlrB(II)) are investigated. The dark-adapted proteins exist in two different receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF(r,f) and BLUF(r,sl)). Some of the flavin-cofactor (ca. 8%) is unbound in thermodynamic equilibrium with the bound cofactor. The two receptor conformations are transformed to putative signalling states (BLUF(s,f) and BLUF(s,sl)) of decreased fluorescence efficiency and shortened fluorescence lifetime by blue-light excitation. In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 2s. Quantum yields of signalling state formation of about 90% for BlrB(II) and about 40% for BlrB(I) were determined by intensity dependent transmission measurements. Extended blue-light excitation causes unbound flavin degradation (formation of lumichrome and lumiflavin-derivatives) and bound cofactor conversion to the semiquinone form. The flavin-semiquinone further reduces and the reduced flavin re-oxidizes back in the dark. A photo-dynamics scheme is presented and relevant quantum efficiencies and time constants are determined.

Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway.

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Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Hunter, C Neil; Bocian, David F; Holten, Dewey; Niedzwiedzki, Dariusz M

2016-06-23

Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC═C + NC═O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids.

Early bacteriopheophytin reduction in charge separation in reaction centers of Rhodobacter sphaeroides.

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Zhu, Jingyi; van Stokkum, Ivo H M; Paparelli, Laura; Jones, Michael R; Groot, Marie Louise

2013-06-04

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on BA and HA via the appearance of the BA and HA anion bands. We observed an unexpectedly early rise of the HA⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between BA and HA, and the effect of protein conformation on the electron transfer rate. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Bioremediation of petroleum hydrocarbon contaminated soil by Rhodobacter sphaeroides biofertilizer and plants.

Science.gov (United States)

Jiao, Haihua; Luo, Jinxue; Zhang, Yiming; Xu, Shengjun; Bai, Zhihui; Huang, Zhanbin

2015-09-01

Bio-augmentation is a promising technique for remediation of polluted soils. This study aimed to evaluate the bio-augmentation effect of Rhodobacter sphaeroides biofertilizer (RBF) on the bioremediation of total petroleum hydrocarbons (TPH) contaminated soil. A greenhouse pot experiment was conducted over a period of 120 days, three methods for enhancing bio-augmentation were tested on TPH contaminated soils, including single addition RBF, planting, and combining of RBF and three crop species, such as wheat (W), cabbage (C) and spinach (S), respectively. The results demonstrated that the best removal of TPH from contaminated soil in the RBF bio-augmentation rhizosphere soils was found to be 46.2%, 65.4%, 67.5% for W+RBF, C+RBF, S+RBF rhizosphere soils respectively. RBF supply impacted on the microbial community diversity (phospholipid fatty acids, PLFA) and the activity of soil enzymes, such as dehydrogenase (DH), alkaline phosphatase (AP) and urease (UR). There were significant difference among the soil only containing crude oil (CK), W, C and S rhizosphere soils and RBF bio-augmentation soils. Moreover, the changes were significantly distinct depended on crops species. It was concluded that the RBF is a valuable material for improving effect of remediation of TPH polluted soils.

Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

International Nuclear Information System (INIS)

Heintz, Udo; Meinhart, Anton; Winkler, Andreas

2014-01-01

Crystal structures of two truncated variants of the transcription factor PpsR from R. sphaeroides are presented that enabled the phasing of a triple PAS domain construct. Together, these structures reveal the importance of α-helical PAS extensions for multi-PAS domain-mediated protein oligomerization and function. Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteins are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsR Q-PAS1 ) and two (PpsR N-Q-PAS1 ) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsR ΔHTH ) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsR ΔHTH structure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N-domain that plays an important

Resonance Raman spectroscopy of 2H-labelled spheroidenes in petroleum ether and in the Rhodobacter sphaeroides reaction centre.

Science.gov (United States)

Kok, P; Köhler, J; Groenen, E J; Gebhard, R; van der Hoef, I; Lugtenburg, J; Farhoosh, R; Frank, H A

1997-03-01

As a step towards the structural analysis of the carotenoid spheroidene in the Rhodobacter sphaeroides reaction centre, we present the resonance Raman spectra of 14-2H, 15-2H, 15'-2H, 14'-2H, 14,15'-2H2 and 15-15'-2H2 spheroidenes in petroleum ether and, except for 14,15'-2H2 spheroidene, in the Rb. sphaeroides R26 reaction center (RC). Analysis of the spectral changes upon isotopic substitution allows a qualitative assignment of most of the vibrational bands to be made. For the all-trans spheroidenes in solution the resonance enhancement of the Raman bands is determined by the participation of carbon carbon stretching modes in the centre of the conjugated chain, the C9 to C15' region. For the RC-bound 15,15'-cis spheroidenes, enhancement is determined by the participation of carbon-carbon stretching modes in the centre of the molecule, the C13 to C13' region. Comparison of the spectra in solution and in the RC reveals evidence for an out-of-plane distortion of the RC-bound spheroidene in the central C14 to C14' region of the carotenoid. The characteristic 1240 cm-1 band in the spectrum of the RC-bound spheroidene has been assigned to a normal mode that contains the coupled C12-C13 and C13'-C12' stretch vibrations.

Study of the selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans

International Nuclear Information System (INIS)

Bernier-Pierru, B.

2006-05-01

Selenium is an essential element for all living organisms at the low level; however it becomes toxic and mutagenic at higher concentrations. The predominant forms of selenium in natural environments are selenate and selenite which are toxic. Bacteria can use several mechanisms of detoxification such as methylation in volatile compounds or reduction in elemental selenium. In this way, our model, Rhodobacter sphaeroides f. sp. denitrificans, is able to reduce selenite into selenium. We have combined biochemical and molecular approaches to better characterize the mechanism and protagonists of this reduction. After studying the physiological response of the bacterium in the presence of selenite, we screened a transposon library in order to isolate mutants with a weakened reduction ability. Two of these selected mutants are affected in genes involved in the molybdenum cofactor synthesis, moaA and mogA. Several reductases, the molybdo enzymes, required this cofactor. Furthermore the addition of tungsten, a competitor for the molybdenum, in the culture medium, dramatically reduces the rate of selenite reduction. These results strongly suggest that a molybdo enzyme is involved in one of the selenite reduction pathways. The potential role of different proteins has been investigated, especially for the nitrate reductase, the DMSO reductase and the biotin sulfoxide reductase. We have also selected a mutant affected in the smoM gene which encodes a peri-plasmic component of a TRAP transporter. The phenotype of this mutant suggests the involvement of this transporter in the selenite import. (author)

Transient absorption study of two-photon excitation mechanism in the LH2 complex from purple bacterium Rhodobacter sphaeroides.

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Stepanenko, Ilya; Kompanetz, Viktor; Makhneva, Zoya; Chekalin, Sergey; Moskalenko, Andrei; Razjivin, Andrei

2012-03-08

The mechanism of two-photon excitation of a peripheral light-harvesting complex LH2 (B800-850) from purple bacterium Rhodobacter sphaeroides was explained on the basis of femtosecond transient absorption data. Fast bleaching of the B850 absorption band was measured under two-photon excitation by 1350 nm femtosecond pulses, showing fast subpicosecond arrival of excitation energy to B850 circular aggregates. Any spectral changes connected with the B800 absorption band of B800-BChl molecules were absent. A similar picture was observed under one-photon excitation of the LH2 complex by 675 nm femtosecond pulses. We believe these effects may be attributed to direct excitation of high-energy excitonic states of a B850 circular aggregate or its vibrational manifold in accordance with the model of Abe [Chem. Phys. 2001, 264, 355-363].

A new nitrilase-producing strain named Rhodobacter sphaeroides LHS-305: biocatalytic characterization and substrate specificity.

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Yang, Chunsheng; Wang, Xuedong; Wei, Dongzhi

2011-12-01

The characteristics of the new nitrilase-producing strain Rhodobacter sphaeroides LHS-305 were investigated. By investigating several parameters influencing nitrilase production, the specific cell activity was ultimately increased from 24.5 to 75.0 μmol g(-1) min(-1), and hereinto, the choice of inducer proved the most important factor. The aromatic nitriles (such as 3-cyanopyridine and benzonitrile) were found to be the most favorable substrates of the nitrilase by analyzing the substrate spectrum. It was speculated that the unsaturated carbon atom attached to the cyano group was crucial for this type of nitrilase. The value of apparent K (m), substrate inhibition constant, and product inhibition constant of the nitrilase against 3-cyanopyridine were 4.5 × 10(-2), 29.2, and 8.6 × 10(-3) mol L(-1), respectively. When applied in nicotinic acid preparation, the nitrilase is able to hydrolyze 200 mmol L(-1) 3-cyanopyridine with 93% conversion rate in 13 h by 6.1 g L(-1) cells (dry cell weight).

Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides

Science.gov (United States)

Zirak, P.; Penzkofer, A.; Schiereis, T.; Hegemann, P.; Jung, A.; Schlichting, I.

2005-08-01

The BLUF domain of the transcriptional anti-repressor protein AppA from the non-sulfur anoxyphototrophic purple bacterium Rhodobacter sphaeroides was characterized by absorption and emission spectroscopy. The BLUF domain constructs AppA 148 (consisting of amino-acid residues 1-148) and AppA 126 (amino-acid residues 1-126) are investigated. The cofactor of the investigated domains is found to consist of a mixture of the flavins riboflavin, FMN, and FAD. The dark-adapted domains exist in two different active receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF r,f and BLUF r,sl) and a small non-interacting conformation (BLUF nc). The active receptor conformations are transformed to putative signalling states (BLUF s,f and BLUF s,sl) of low fluorescence efficiency and picosecond fluorescence lifetime by blue-light excitation (light-adapted domains). In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 17 min. A quantum yield of signalling state formation of about 25% was determined by intensity dependent transmission measurements. A photo-cycle scheme is presented including photo-induced charge transfer complex formation, charge recombination, and protein binding pocket reorganisation.

Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides

International Nuclear Information System (INIS)

Zirak, P.; Penzkofer, A.; Schiereis, T.; Hegemann, P.; Jung, A.; Schlichting, I.

2005-01-01

The BLUF domain of the transcriptional anti-repressor protein AppA from the non-sulfur anoxyphototrophic purple bacterium Rhodobacter sphaeroides was characterized by absorption and emission spectroscopy. The BLUF domain constructs AppA 148 (consisting of amino-acid residues 1-148) and AppA 126 (amino-acid residues 1-126) are investigated. The cofactor of the investigated domains is found to consist of a mixture of the flavins riboflavin, FMN, and FAD. The dark-adapted domains exist in two different active receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF r,f and BLUF r,sl ) and a small non-interacting conformation (BLUF nc ). The active receptor conformations are transformed to putative signalling states (BLUF s,f and BLUF s,sl ) of low fluorescence efficiency and picosecond fluorescence lifetime by blue-light excitation (light-adapted domains). In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 17 min. A quantum yield of signalling state formation of about 25% was determined by intensity dependent transmission measurements. A photo-cycle scheme is presented including photo-induced charge transfer complex formation, charge recombination, and protein binding pocket reorganisation

A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization.

Science.gov (United States)

Wang, Hualei; Li, Guinan; Li, Mingyang; Wei, Dongzhi; Wang, Xuedong

2014-01-01

In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V max and K m toward 3-cyanopyridine were 77.5 μmol min(-1) mg(-1) and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.

Structure and function of cytochrome c2 in electron transfer complexes with the photosynthetic reaction center of Rhodobacter sphaeroides: optical linear dichroism and EPR.

Science.gov (United States)

Drepper, F; Mathis, P

1997-02-11

The photosynthetic reaction center (RC) and its secondary electron donor the water-soluble cytochrome (cyt) c2 from the purple bacterium Rhodobacter sphaeroides have been used in cross-linked and non-cross-linked complexes, oriented in compressed gels or partially dried multilayers, to study the respective orientation of the primary donor P (BChl dimer) and of cyt c2. Three methods were used: (i) Polarized optical absorption spectra at 295 and 10 K were measured and the linear dichroism of the two individual transitions (Qx, Qy), which are nearly degenerate within the alpha-band of reduced cyt c2, was determined. Attribution of the polarization directions to the molecular axes within the heme plane yielded the average cyt orientation in the complexes. (ii) Time-resolved flash absorption measurements using polarized light allowed determination of the orientation of cyt c2 in complexes which differ in their kinetics of electron transfer. (iii) EPR spectroscopy of ferricyt c2 in cross-linked RC-cyt c2 complexes was used to determine the angle between the heme and the membrane plane. The results suggest the following structural properties for the docking of cyt c2 to the RC: (i) In cross-linked complexes, the two cytochromes displaying half-lives of 0.7 and 60 micros for electron transfer to P+ are similarly oriented (difference plane is parallel to the symmetry axis of the RC (0 degrees +/- 10 degrees). Moreover, the Qy transition, which is assumed to be polarized within the ring III-ring I direction of the heme plane, makes an angle of 56 degrees +/- 1 degree with the symmetry axis. (iii) The dichroism spectrum for the fast phase (0.7 micros) for the non-cross-linked cyt c2-RC complex suggests an orientation similar to that of cross-linked cyt c2, but the heme plane is tilted about 20 degrees closer to the membrane. An alternative model is that two or more bound states of cyt c2 with heme plane tilt angles between 0 degrees and 30 degrees allow the fast electron

Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides

Energy Technology Data Exchange (ETDEWEB)

Zirak, P. [Institut II - Experimentelle und Angewandte Physik, Universitaet Regensburg, Universitaetstrasse 31, D-93053 Regensburg (Germany); Penzkofer, A. [Institut II - Experimentelle und Angewandte Physik, Universitaet Regensburg, Universitaetstrasse 31, D-93053 Regensburg (Germany)], E-mail: alfons.penzkofer@physik.uni-regensburg.de; Schiereis, T. [Institut fuer Biologie, Experimentelle Biophysik, Humboldt-Universitaet zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany); Hegemann, P. [Institut fuer Biologie, Experimentelle Biophysik, Humboldt-Universitaet zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany); Jung, A. [Max-Planck-Institut fuer medizinische Forschung, Abteilung Biomolekulare Mechanismen, Jahnstrasse 29, D-69120 Heidelberg (Germany); Schlichting, I. [Max-Planck-Institut fuer medizinische Forschung, Abteilung Biomolekulare Mechanismen, Jahnstrasse 29, D-69120 Heidelberg (Germany)

2005-08-08

The BLUF domain of the transcriptional anti-repressor protein AppA from the non-sulfur anoxyphototrophic purple bacterium Rhodobacter sphaeroides was characterized by absorption and emission spectroscopy. The BLUF domain constructs AppA{sub 148} (consisting of amino-acid residues 1-148) and AppA{sub 126} (amino-acid residues 1-126) are investigated. The cofactor of the investigated domains is found to consist of a mixture of the flavins riboflavin, FMN, and FAD. The dark-adapted domains exist in two different active receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF{sub r,f} and BLUF{sub r,sl}) and a small non-interacting conformation (BLUF{sub nc}). The active receptor conformations are transformed to putative signalling states (BLUF{sub s,f} and BLUF{sub s,sl}) of low fluorescence efficiency and picosecond fluorescence lifetime by blue-light excitation (light-adapted domains). In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 17 min. A quantum yield of signalling state formation of about 25% was determined by intensity dependent transmission measurements. A photo-cycle scheme is presented including photo-induced charge transfer complex formation, charge recombination, and protein binding pocket reorganisation.

Direct visualization of exciton reequilibration in the LH1 and LH2 complexes of Rhodobacter sphaeroides by multipulse spectroscopy.

Science.gov (United States)

Cohen Stuart, Thomas A; Vengris, Mikas; Novoderezhkin, Vladimir I; Cogdell, Richard J; Hunter, C Neil; van Grondelle, Rienk

2011-05-04

The dynamics of the excited states of the light-harvesting complexes LH1 and LH2 of Rhodobacter sphaeroides are governed, mainly, by the excitonic nature of these ring-systems. In a pump-dump-probe experiment, the first pulse promotes LH1 or LH2 to its excited state and the second pulse dumps a portion of the excited state. By selective dumping, we can disentangle the dynamics normally hidden in the excited-state manifold. We find that by using this multiple-excitation technique we can visualize a 400-fs reequilibration reflecting relaxation between the two lowest exciton states that cannot be directly explored by conventional pump-probe. An oscillatory feature is observed within the exciton reequilibration, which is attributed to a coherent motion of a vibrational wavepacket with a period of ∼150 fs. Our disordered exciton model allows a quantitative interpretation of the observed reequilibration processes occurring in these antennas. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using a novel strategy - shaking and extra-light supplementation approach

Energy Technology Data Exchange (ETDEWEB)

Li, Xu; Wang, Yong-Hong; Zhang, Si-Liang; Chu, Ju; Zhang, Ming; Huang, Ming-Zhi; Zhuang, Ying-Ping [State Key Laboratory of Bioreactor Engineering, P.O. Box 329, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237 (China)

2009-12-15

Biohydrogen has gained attention due to its potential as a sustainable alternative to conventional methods for hydrogen production. In this study, the effect of light intensity as well as cultivation method (standing- and shaking-culture) on the cell growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated in 38-ml anaerobic photobioreactor with RCVBN medium. Thus, a novel shaking and extra-light supplementation (SELS) approach was developed to enhance the phototrophic H{sub 2} production by R. sphaeroides ZX-5 using malate as the sole carbon source. The optimum illumination condition for shaking-culture by strain ZX-5 increased to 7000-8000 lux, markedly higher than that for standing-culture (4000-5000 lux). Under shaking and elevated illumination (7000-8000 lux), the culture was effective in promoting photo-H{sub 2} production, resulting in a 59% and 56% increase of the maximum and average hydrogen production rate, respectively, in comparison with the culture under standing and 4000-5000 lux conditions. The highest hydrogen-producing rate of 165.9 ml H{sub 2}/l h was observed under the application of SELS approach. To our knowledge, this record is currently the highest hydrogen production rate of non-immobilized purple non-sulphur (PNS) bacteria. This optimal performance of photo-H{sub 2} production using SELS approach is a favorable choice of sustainable and economically feasible strategy to improve phototrophic H{sub 2} production efficiency. (author)

Redox potential tuning through differential quinone binding in the photosynthetic reaction center of Rhodobacter sphaeroides.

Science.gov (United States)

Vermaas, Josh V; Taguchi, Alexander T; Dikanov, Sergei A; Wraight, Colin A; Tajkhorshid, Emad

2015-03-31

Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force field and subsequent molecular dynamics simulations of the quinone-bound RC, we have investigated and characterized the interactions of the protein with the quinones in the Q(A) and Q(B) sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the Q(B) site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential difference between the quinones occupying the Q(A) and Q(B) sites. Disruption of this interaction leads to weaker binding in a ubiquinone analogue that lacks a 2-methoxy group, a finding supported by reverse electron transfer electron paramagnetic resonance experiments of the Q(A)⁻Q(B)⁻ biradical and competitive binding assays.

Role of PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 1. PufX is required for efficient light-driven electron transfer and photophosphorylation under anaerobic conditions.

Science.gov (United States)

Barz, W P; Francia, F; Venturoli, G; Melandri, B A; Verméglio, A; Oesterhelt, D

1995-11-21

The pufX gene is essential for photoheterotrophic growth of the purple bacterium Rhodobacter sphaeroides. In order to analyze the molecular function of the PufX membrane protein, we constructed a chromosomal pufX deletion mutant and phenotypically compared it to a pufX+ control strain and to two suppressor mutants which are able to grow photosynthetically in the absence of pufX. Using this genetic background, we confirmed that PufX is required for photoheterotrophic growth under anaerobic conditions, although all components of the photosynthetic apparatus were present in similar amounts in all strains investigated. We show that the deletion of PufX is not lethal for illuminated pufX- cells, suggesting that PufX is required for photosynthetic cell division. Since chromatophores isolated from the pufX- mutant were found to be unsealed vesicles, the role of PufX in photosynthetic energy transduction was studied in vivo. We show that PufX is essential for light-induced ATP synthesis (photophosphorylation) in anaerobically incubated cells. Measurements of absorption changes induced by a single turnover flash demonstrated that PufX is not required for electron flow through the reaction center and the cytochrome bc1 complex under anaerobic conditions. During prolonged illumination, however, PufX is essential for the generation of a sufficiently large membrane potential to allow photosynthetic growth. These in vivo results demonstrate that under anaerobic conditions PufX plays an essential role in facilitating effective interaction of the components of the photosynthetic apparatus.

Effect of co-substrate on production of poly-β- hydroxybutyrate (PHB and copolymer PHBV from newly identified mutant Rhodobacter sphaeroides U7 cultivated under aerobic-dark condition

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Kemarajt Kemavongse

2007-07-01

Full Text Available Photosynthetic bacterial mutant strain U7 was identified using both classical and molecular (16S rDNA techniques to be Rhodobacter sphaeroides. The glutamate-acetate (GA medium containing sodium acetate and sodium glutamate as carbon and nitrogen sources was used for production of poly-β-hydroxybutyrate (PHB from R. sphaeroides U7 cultivated under aerobic-dark condition (200 rpm at 37oC. Effect of auxiliary carbon sources (propionate and valerate and concentrations (molar ratio of 40/0, 40/20, 40/40 and 40/80 on copolymer production were studied. Both combinations of acetate with valerate and acetate with propionate were found to induce the accumulation of poly-β-hydroxybutyrate-co-β-hydroxyvalerate (PHBV within the cell. Acetate with propionate in the molar ratio of 40/40 gave the highest poly-β-hydroxyalkanoates (PHA content (77.68%, followed by acetate with valerate at the same molar ratio (77.42%. Although their polymer contents were similar, the presence of 40 mM valerate gave more than 4 times higher hydroxyvalerate (HV fraction (84.77% than in the presence of 40 mM propionate (19.12% HV fraction.

Elimination of polarity in the carotenoid terminus promotes the exposure of B850-binding sites (Tyr 44, 45) and ANS-mediated energy transfer in LH2 complexes of Rhodobacter sphaeroides.

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Liu, Yuan; Wu, Yongqiang; Xu, Chunhe

2004-12-10

Carotenoids in the peripheral light-harvesting complexes (LH2) of the green mutant (GM309) of Rhodobacter sphaeroides were identified as containing neurosporenes, which lack the polar CH(3)O group, compared to spheroidenes in native-LH2 of R. sphaeroides 601. After LH2 complexes were treated with 1-anilino-8-naphthalene sulfonate (ANS), new energy transfer pathways from ANS or tryptophan to carotenoids were discovered in both native- and GM309-LH2. The carotenoid fluorescence intensity of GM309-LH2 was greater than that of native-LH2 when bound with ANS, suggesting that the elimination of polarity in the neurosporene increases the energy transfer from ANS to carotenoid. The fact that two alpha-tyrosines (alpha-Tyr 44, 45, B850-binding sites) in each alpha-apoprotein of GM309-LH2 were more easily modified than those of native-LH2 by N-acetylimidazole (NAI) indicates that the elimination of polarity in the neurosporene terminus increases the exposure of these sites to solution.

Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c.

Science.gov (United States)

Battistuzzi, G; Borsari, M; Sola, M; Francia, F

1997-12-23

The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

The mutation of carotenoids affects the energy transfer in LH2 light harvesting complexes from Rhodobacter sphaeroides 601 at room temperature

International Nuclear Information System (INIS)

Liu Weimin; Liu Yuan; Guo Lijun; Xu Chunhe; Qian Shixiong

2006-01-01

Energy transfer in two kinds of peripheral antenna complexes LH2 from Rhodobacter sphaeroides 601 was studied by absorption, fluorescence emission, time-resolved fluorescence and femtosecond transient absorption spectroscopy at room temperature. These two complexes are LH2 (RS601) and green carotenoid mutated LH2 (GM309). The obtained results demonstrate that: (a) compared with spheroidenes, which have ten carbon-carbon double bonds in native RS601, carotenoids in GM309 were identified as containing neurosporenes with nine carbon-carbon double bonds, which show a significant blue shift of ∼20 nm in the three absorption peaks because of the higher energy levels of neurosporene than those of spheroidene, (b) the higher energy levels of neurosporene in GM309 induce a lower B800 → B850 energy transfer rate and efficiency as compared to that in RS601 resulting from the relatively higher band gap between the donor of B800 and the bridge of the carotenoids (c) the same lifetime of the B850 excited singlet state is observed in these two LH2 complexes

Photo dynamics of BLUF domain mutant H44R of AppA from Rhodobacter sphaeroides

International Nuclear Information System (INIS)

Zirak, P.; Penzkofer, A.; Hegemann, P.; Mathes, T.

2007-01-01

The photo-cycle dynamics of the H44R mutant of the BLUF domain of the transcriptional anti-repressor protein AppA (AppA-H44R) from the non-sulfur anoxyphototropic purple bacterium Rhodobacter sphaeroides is studied in order to gain information on the involvement of His44 in the photo-cyclic mechanism of the AppA BLUF domain and to add information to the involved processes. The amino acid residue histidine at position 44 is replaced by arginine. A 12 nm red-shifted signalling state is formed upon blue-light excitation, while in wild-type AppA (AppA-wt) the red-shift is 16 nm. The recovery to the receptor dark state is approximately a factor of 2.5 faster (τ rec ∼ 6.5 min) than the recovery of the wild-type counterpart. Extended light exposure of the mutant causes photo-degradation of flavin (mainly free flavin conversion to lumichrome and re-equilibration between free and non-covalently bound flavin) and protein aggregation (showing up as light scattering). No photo-degradation was observed for AppA-wt. The quantum efficiency of signalling-state formation determined by intensity dependent absorption measurements is found to be φ s ∼ 0.3 (for AppA-wt: φ s ∼ 0.24). A two-component single-exponential fluorescence relaxation was observed, which is interpreted as fast fluorescence quenching to an equilibrium value by photo-induced electron transfer followed by slower fluorescence decay due to charge recombination. Based on the experimental findings, an extended photo-cycle model for BLUF domains is proposed

Photo dynamics of BLUF domain mutant H44R of AppA from Rhodobacter sphaeroides

Science.gov (United States)

Zirak, P.; Penzkofer, A.; Hegemann, P.; Mathes, T.

2007-05-01

The photo-cycle dynamics of the H44R mutant of the BLUF domain of the transcriptional anti-repressor protein AppA (AppA-H44R) from the non-sulfur anoxyphototropic purple bacterium Rhodobacter sphaeroides is studied in order to gain information on the involvement of His44 in the photo-cyclic mechanism of the AppA BLUF domain and to add information to the involved processes. The amino acid residue histidine at position 44 is replaced by arginine. A 12 nm red-shifted signalling state is formed upon blue-light excitation, while in wild-type AppA (AppA-wt) the red-shift is 16 nm. The recovery to the receptor dark state is approximately a factor of 2.5 faster ( τrec ≈ 6.5 min) than the recovery of the wild-type counterpart. Extended light exposure of the mutant causes photo-degradation of flavin (mainly free flavin conversion to lumichrome and re-equilibration between free and non-covalently bound flavin) and protein aggregation (showing up as light scattering). No photo-degradation was observed for AppA-wt. The quantum efficiency of signalling-state formation determined by intensity dependent absorption measurements is found to be ϕs ≈ 0.3 (for AppA-wt: ϕs ≈ 0.24). A two-component single-exponential fluorescence relaxation was observed, which is interpreted as fast fluorescence quenching to an equilibrium value by photo-induced electron transfer followed by slower fluorescence decay due to charge recombination. Based on the experimental findings, an extended photo-cycle model for BLUF domains is proposed.

Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MS(n)).

Science.gov (United States)

Granafei, Sara; Losito, Ilario; Trotta, Massimo; Italiano, Francesca; de Leo, Vincenzo; Agostiano, Angela; Palmisano, Francesco; Cataldi, Tommaso R I

2016-01-15

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MS(n), n = 2-4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M-H](-) at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M-H](-) ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic

Probing energy transfer events in the light harvesting complex 2 (LH2) of Rhodobacter sphaeroides with two-dimensional spectroscopy.

Science.gov (United States)

Fidler, Andrew F; Singh, Ved P; Long, Phillip D; Dahlberg, Peter D; Engel, Gregory S

2013-10-21

Excitation energy transfer events in the photosynthetic light harvesting complex 2 (LH2) of Rhodobacter sphaeroides are investigated with polarization controlled two-dimensional electronic spectroscopy. A spectrally broadened pulse allows simultaneous measurement of the energy transfer within and between the two absorption bands at 800 nm and 850 nm. The phased all-parallel polarization two-dimensional spectra resolve the initial events of energy transfer by separating the intra-band and inter-band relaxation processes across the two-dimensional map. The internal dynamics of the 800 nm region of the spectra are resolved as a cross peak that grows in on an ultrafast time scale, reflecting energy transfer between higher lying excitations of the B850 chromophores into the B800 states. We utilize a polarization sequence designed to highlight the initial excited state dynamics which uncovers an ultrafast transfer component between the two bands that was not observed in the all-parallel polarization data. We attribute the ultrafast transfer component to energy transfer from higher energy exciton states to lower energy states of the strongly coupled B850 chromophores. Connecting the spectroscopic signature to the molecular structure, we reveal multiple relaxation pathways including a cyclic transfer of energy between the two rings of the complex.

Probing energy transfer events in the light harvesting complex 2 (LH2) of Rhodobacter sphaeroides with two-dimensional spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Fidler, Andrew F.; Singh, Ved P.; Engel, Gregory S. [Department of Chemistry, The Institute for Biophysical Dynamics, and The James Franck Institute, The University of Chicago, Chicago, Illinois 60637 (United States); Long, Phillip D.; Dahlberg, Peter D. [Graduate Program in the Biophysical Sciences, The University of Chicago, Chicago, Illinois 60637 (United States)

2013-10-21

Excitation energy transfer events in the photosynthetic light harvesting complex 2 (LH2) of Rhodobacter sphaeroides are investigated with polarization controlled two-dimensional electronic spectroscopy. A spectrally broadened pulse allows simultaneous measurement of the energy transfer within and between the two absorption bands at 800 nm and 850 nm. The phased all-parallel polarization two-dimensional spectra resolve the initial events of energy transfer by separating the intra-band and inter-band relaxation processes across the two-dimensional map. The internal dynamics of the 800 nm region of the spectra are resolved as a cross peak that grows in on an ultrafast time scale, reflecting energy transfer between higher lying excitations of the B850 chromophores into the B800 states. We utilize a polarization sequence designed to highlight the initial excited state dynamics which uncovers an ultrafast transfer component between the two bands that was not observed in the all-parallel polarization data. We attribute the ultrafast transfer component to energy transfer from higher energy exciton states to lower energy states of the strongly coupled B850 chromophores. Connecting the spectroscopic signature to the molecular structure, we reveal multiple relaxation pathways including a cyclic transfer of energy between the two rings of the complex.

Probing energy transfer events in the light harvesting complex 2 (LH2) of Rhodobacter sphaeroides with two-dimensional spectroscopy

International Nuclear Information System (INIS)

Fidler, Andrew F.; Singh, Ved P.; Engel, Gregory S.; Long, Phillip D.; Dahlberg, Peter D.

2013-01-01

Excitation energy transfer events in the photosynthetic light harvesting complex 2 (LH2) of Rhodobacter sphaeroides are investigated with polarization controlled two-dimensional electronic spectroscopy. A spectrally broadened pulse allows simultaneous measurement of the energy transfer within and between the two absorption bands at 800 nm and 850 nm. The phased all-parallel polarization two-dimensional spectra resolve the initial events of energy transfer by separating the intra-band and inter-band relaxation processes across the two-dimensional map. The internal dynamics of the 800 nm region of the spectra are resolved as a cross peak that grows in on an ultrafast time scale, reflecting energy transfer between higher lying excitations of the B850 chromophores into the B800 states. We utilize a polarization sequence designed to highlight the initial excited state dynamics which uncovers an ultrafast transfer component between the two bands that was not observed in the all-parallel polarization data. We attribute the ultrafast transfer component to energy transfer from higher energy exciton states to lower energy states of the strongly coupled B850 chromophores. Connecting the spectroscopic signature to the molecular structure, we reveal multiple relaxation pathways including a cyclic transfer of energy between the two rings of the complex

Photo dynamics of BLUF domain mutant H44R of AppA from Rhodobacter sphaeroides

Energy Technology Data Exchange (ETDEWEB)

Zirak, P. [Institut II - Experimentelle und Angewandte Physik, Universitaet Regensburg, Universitaetstrasse 31, D-93053 Regensburg (Germany); Penzkofer, A. [Institut II - Experimentelle und Angewandte Physik, Universitaet Regensburg, Universitaetstrasse 31, D-93053 Regensburg (Germany)], E-mail: alfons.penzkofer@physik.uni-regensburg.de; Hegemann, P.; Mathes, T. [Institut fuer Biologie, Experimentelle Biophysik, Humboldt-Universitaet zu Berlin, Invalidenstr. 42, D-10115 Berlin (Germany)

2007-05-21

The photo-cycle dynamics of the H44R mutant of the BLUF domain of the transcriptional anti-repressor protein AppA (AppA-H44R) from the non-sulfur anoxyphototropic purple bacterium Rhodobacter sphaeroides is studied in order to gain information on the involvement of His44 in the photo-cyclic mechanism of the AppA BLUF domain and to add information to the involved processes. The amino acid residue histidine at position 44 is replaced by arginine. A 12 nm red-shifted signalling state is formed upon blue-light excitation, while in wild-type AppA (AppA-wt) the red-shift is 16 nm. The recovery to the receptor dark state is approximately a factor of 2.5 faster ({tau}{sub rec} {approx} 6.5 min) than the recovery of the wild-type counterpart. Extended light exposure of the mutant causes photo-degradation of flavin (mainly free flavin conversion to lumichrome and re-equilibration between free and non-covalently bound flavin) and protein aggregation (showing up as light scattering). No photo-degradation was observed for AppA-wt. The quantum efficiency of signalling-state formation determined by intensity dependent absorption measurements is found to be {phi}{sub s} {approx} 0.3 (for AppA-wt: {phi}{sub s} {approx} 0.24). A two-component single-exponential fluorescence relaxation was observed, which is interpreted as fast fluorescence quenching to an equilibrium value by photo-induced electron transfer followed by slower fluorescence decay due to charge recombination. Based on the experimental findings, an extended photo-cycle model for BLUF domains is proposed.

The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate

Czech Academy of Sciences Publication Activity Database

Sjöholm, C.; Bergstrand, J.; Nilsson, T.; Šachl, Radek; von Ballmoos, Ch.; Widengren, J.; Brzezinski, P.

2017-01-01

Roč. 7, č. 1 (2017), č. článku 2926. ISSN 2045-2322 Institutional support: RVO:61388955 Keywords : biological energy -conversion * cytochrome-c-oxidase * membrane-surface * rhodobacter-sphaeroides Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 4.259, year: 2016

Functional characteristics of spirilloxanthin and keto-bearing Analogues in light-harvesting LH2 complexes from Rhodobacter sphaeroides with a genetically modified carotenoid synthesis pathway.

Science.gov (United States)

Niedzwiedzki, Dariusz M; Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Bocian, David F; Holten, Dewey; Hunter, C Neil

2015-01-01

Light-harvesting 2 (LH2) complexes from a genetically modified strain of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. Carotenoid synthesis in the Rba. sphaeroides strain was engineered to redirect carotenoid production away from spheroidene into the spirilloxanthin synthesis pathway. The strain assembles LH2 antennas with substantial amounts of spirilloxanthin (total double-bond conjugation length N=13) if grown anaerobically and of keto-bearing long-chain analogs [2-ketoanhydrorhodovibrin (N=13), 2-ketospirilloxanthin (N=14) and 2,2'-diketospirilloxanthin (N=15)] if grown semi-aerobically (with ratios that depend on growth conditions). We present the photophysical, electronic, and vibrational properties of these carotenoids, both isolated in organic media and assembled within LH2 complexes. Measurements of excited-state energy transfer to the array of excitonically coupled bacteriochlorophyll a molecules (B850) show that the mean lifetime of the first singlet excited state (S1) of the long-chain (N≥13) carotenoids does not change appreciably between organic media and the protein environment. In each case, the S1 state appears to lie lower in energy than that of B850. The energy-transfer yield is ~0.4 in LH2 (from the strain grown aerobically or semi-aerobically), which is less than half that achieved for LH2 that contains short-chain (N≤11) analogues. Collectively, the results suggest that the S1 excited state of the long-chain (N≥13) carotenoids participates little if at all in carotenoid-to-BChl a energy transfer, which occurs predominantly via the carotenoid S2 excited state in these antennas. Copyright © 2015 Elsevier B.V. All rights reserved.

Exploitation of dark fermented effluent of cheese whey by co-culture of Rhodobacter sphaeroides and Bacillus firmus for photo-hydrogen production.

Science.gov (United States)

Pandey, A; Pandey, A

2017-07-31

In this study photo-hydrogen production from cheese whey dark fermentation (DF) effluent by the co-culture of Rhodobacter sphaeroides -NMBL-01 and Bacillus firmus - NMBL-03 has been reported. The effect of pH, initial chemical oxygen demand (COD) and the concentration effect of FeSO4.7H2O on photo-hydrogen production have been investigated. The end products of dark fermentation effluent of cheese whey were mainly comprised of soluble organic acids, i.e. butyric acid and lactic acid. The batch process was carried out under light intensity of 2.5 kLux at 32 ± 2oC without any addition of extra carbon and nitrogen source. The single parameter optimization studies revealed optimum pH 6.5, initial COD 4.71 g/L and supplementation of Fe2+ concentration 100 mg/L. The maximum cumulative hydrogen production and yield were found to be 469 ± 45.8 ml H2/L and 146.56 ± 14.31 ml H2/g COD reduced (67.9% reduction in COD) respectively. The mutual interactions among the process parameters were also investigated by three factorial Box-Behnken design of response surface methodology. The optimized experimental values were found concurrent with the calculated values obtained from the theoretical model.

Grain dust-induced lung inflammation is reduced by Rhodobacter sphaeroides diphosphoryl lipid A.

Science.gov (United States)

Jagielo, P J; Quinn, T J; Qureshi, N; Schwartz, D A

1998-01-01

To further determine the importance of endotoxin in grain dust-induced inflammation of the lower respiratory tract, we evaluated the efficacy of pentaacylated diphosphoryl lipid A derived from the lipopolysaccharide of Rhodobacter sphaeroides (RsDPLA) as a partial agonist of grain dust-induced airway inflammation. RsDPLA is a relatively inactive compound compared with lipid A derived from Escherichia coli (LPS) and has been demonstrated to act as a partial agonist of LPS-induced inflammation. To assess the potential stimulatory effect of RsDPLA in relation to LPS, we incubated THP-1 cells with RsDPLA (0.001-100 micrograms/ml), LPS (0.02 microgram endotoxin activity/ml), or corn dust extract (CDE; 0.02 microgram endotoxin activity/ml). Incubation with RsDPLA revealed a tumor necrosis factor (TNF)-alpha stimulatory effect at 100 micrograms/ml. In contrast, incubation with LPS or CDE resulted in TNF-alpha release at 0.02 microgram/ml. Pretreatment of THP-1 cells with varying concentrations of RsDPLA before incubation with LPS or CDE (0.02 microgram endotoxin activity/ml) resulted in a dose-dependent reduction in the LPS- or CDE-induced release of TNF-alpha with concentrations of RsDPLA of up to 10 micrograms/ml but not at 100 micrograms/ml. To further understand the role of endotoxin in grain dust-induced airway inflammation, we utilized the unique LPS inhibitory property of RsDPLA to determine the inflammatory response to inhaled CDE in mice in the presence of RsDPLA. Ten micrograms of RsDPLA intratracheally did not cause a significant inflammatory response compared with intratracheal saline. However, pretreatment of mice with 10 micrograms of RsDPLA intratracheally before exposure to CDE (5.4 and 0.2 micrograms/m3) or LPS (7.2 and 0.28 micrograms/m3) resulted in significant reductions in the lung lavage concentrations of total cells, neutrophils, and specific proinflammatory cytokines compared with mice pretreated with sterile saline. These results confirm the LPS

Chronic Exposure to Rhodobacter Sphaeroides Extract Lycogen™ Prevents UVA-Induced Malondialdehyde Accumulation and Procollagen I Down-Regulation in Human Dermal Fibroblasts

Science.gov (United States)

Yang, Tsai-Hsiu; Lai, Ying-Hsiu; Lin, Tsuey-Pin; Liu, Wen-Sheng; Kuan, Li-Chun; Liu, Chia-Chyuan

2014-01-01

UVA contributes to the pathogenesis of skin aging by downregulation of procollagen I content and induction of matrix metalloproteinase (MMP)-associated responses. Application of antioxidants such as lycopene has been demonstrated as a convenient way to achieve protection against skin aging. Lycogen™, derived from the extracts of Rhodobacter sphaeroides, exerts several biological effects similar to that of lycopene whereas most of its anti-aging efficacy remains uncertain. In this study, we attempted to examine whether Lycogen™ could suppress malondialdehyde (MDA) accumulation and restore downregulated procollagen I expression induced by UVA exposure. In human dermal fibroblasts Hs68 cells, UVA repressed cell viability and decreased procollagen I protein content accompanied with the induction of MMP-1 and MDA accumulation. Remarkably, incubation with 50 μM Lycogen™ for 24 h ameliorated UVA-induced cell death and restored UVA-induced downregulation of procollagen in a dose-related manner. Lycogen™ treatment also prevented the UVA-induced MMP-1 upregulation and intracellular MDA generation in Hs68 cells. Activation of NFκB levels, one of the downstream events induced by UVA irradiation and MMP-1 induction, were also prevented by Lycogen™ administration. Taken together, our findings demonstrate that Lycogen™ may be an alternative agent that prevents UVA-induced skin aging and could be used in cosmetic and pharmaceutical applications. PMID:24463291

Biochemical analysis and the preliminary crystallographic characterization of D-tagatose 3-epimerase from Rhodobacter sphaeroides.

Science.gov (United States)

Qi, Zhengliang; Zhu, Zhangliang; Wang, Jian-Wen; Li, Songtao; Guo, Qianqian; Xu, Panpan; Lu, Fuping; Qin, Hui-Min

2017-11-09

D-Tagatose 3-epimerase epimerizes D-fructose to yield D-psicose, which is a rare sugar that exists in small quantities in nature and is difficult to synthesize chemically. We aim to explore potential industrial biocatalysts for commercial-scale manufacture of this rare sugar. A D-tagatose 3-epimerase from Rhodobacter sphaeroides (RsDTE) has recently been identified as a D-tagatose 3-epimerase that can epimerize D-fructose to yield D-psicose with a high conversion rate. The purified RsDTE by Ni-affinity chromatography, ionic exchange chromatography and gel filtration forms a tetramer in solution. The maximal activity was in Tris-HCl buffer pH 8.5, and the optimal temperature was at 35 °C. The product, D-psicose, was confirmed using HPLC and NMR. Crystals of RsDTE were obtained using crystal kits and further refined under crystallization conditions such as 10% PEG 8000,0.1 M HEPES pH 7.5, and 8% ethylene glycol at 20 °C using the sitting-drop vapor diffusion method. The RsDTE homology model showed that it possessed the characteristic TIM-barrel fold. Four residues, Glu156, Asp189, Gln215 and Glu250, forms a hydrogen bond network with the active Mn(II) for the hydride transfer reaction. These residues may constitute the catalytic tetrad of RsDTE. The residues around O1, O2 and O3 of the substrates were conserved. However, the binding-site residues are different at O4, O5 and O6. Arg118 formed the unique hydrogen bond with O4 of D-fructose which indicates RsDTE's preference of D-fructose more than any other family enzymes. RsDTE possesses a different metal-binding site. Arg118, forming unique hydrogen bond with O4 of D-fructose, regulates the substrate recognition. The research on D-tagatose 3-epimerase or D-psicose 3-epimerase enzymes attracts enormous commercial interest and would be widely used for rare sugar production in the future.

NCBI nr-aa BLAST: CBRC-RMAC-01-0051 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-RMAC-01-0051 ref|YP_001033817.1| Ice nucleation protein [Rhodobacter sphaeroid...es 2.4.1] gb|ABA81712.1| Ice nucleation protein [Rhodobacter sphaeroides 2.4.1] YP_001033817.1 0.016 26% ...

NCBI nr-aa BLAST: CBRC-OLAT-26-0102 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-OLAT-26-0102 ref|YP_001033817.1| Ice nucleation protein [Rhodobacter sphaeroid...es 2.4.1] gb|ABA81712.1| Ice nucleation protein [Rhodobacter sphaeroides 2.4.1] YP_001033817.1 0.45 25% ...

NCBI nr-aa BLAST: CBRC-XTRO-01-0470 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-XTRO-01-0470 ref|YP_001033817.1| Ice nucleation protein [Rhodobacter sphaeroid...es 2.4.1] gb|ABA81712.1| Ice nucleation protein [Rhodobacter sphaeroides 2.4.1] YP_001033817.1 1e-12 25% ...

NCBI nr-aa BLAST: CBRC-XTRO-01-0267 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-XTRO-01-0267 ref|YP_001033817.1| Ice nucleation protein [Rhodobacter sphaeroid...es 2.4.1] gb|ABA81712.1| Ice nucleation protein [Rhodobacter sphaeroides 2.4.1] YP_001033817.1 6e-27 25% ...

NCBI nr-aa BLAST: CBRC-TNIG-22-0265 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-TNIG-22-0265 ref|YP_001033817.1| Ice nucleation protein [Rhodobacter sphaeroid...es 2.4.1] gb|ABA81712.1| Ice nucleation protein [Rhodobacter sphaeroides 2.4.1] YP_001033817.1 4e-36 37% ...

NCBI nr-aa BLAST: CBRC-OGAR-01-0310 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-OGAR-01-0310 ref|YP_001033817.1| Ice nucleation protein [Rhodobacter sphaeroid...es 2.4.1] gb|ABA81712.1| Ice nucleation protein [Rhodobacter sphaeroides 2.4.1] YP_001033817.1 4e-07 24% ...

Effects of the cryptochrome CryB from Rhodobacter sphaeroides on global gene expression in the dark or blue light or in the presence of singlet oxygen.

Directory of Open Access Journals (Sweden)

Sebastian Frühwirth

Full Text Available Several regulators are controlling the formation of the photosynthetic apparatus in the facultatively photosynthetic bacterium Rhodobacter sphaeroides. Among the proteins affecting photosynthesis gene expression is the blue light photoreceptor cryptochrome CryB. This study addresses the effect of CryB on global gene expression. The data reveal that CryB does not only influence photosynthesis gene expression but also genes for the non-photosynthetic energy metabolism like citric acid cycle and oxidative phosphorylation. In addition several genes involved in RNA processing and in transcriptional regulation are affected by a cryB deletion. Although CryB was shown to undergo a photocycle it does not only affect gene expression in response to blue light illumination but also in response to singlet oxygen stress conditions. While there is a large overlap in these responses, some CryB-dependent effects are specific for blue-light or photooxidative stress. In addition to protein-coding genes some genes for sRNAs show CryB-dependent expression. These findings give new insight into the function of bacterial cryptochromes and demonstrate for the first time a function in the oxidative stress response.

Hydrogen production by using Rhodobacter capsulatus mutants with genetically modified electron transfer chains

Energy Technology Data Exchange (ETDEWEB)

OEztuerk, Yavuz; Yuecel, Meral; Guenduez, Ufuk [Department of Biology, Middle East Technical University, Ankara (Turkey); Daldal, Fevzi [Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, PA 19104-6018 (United States); Mandaci, Sevnur [TUEBITAK Research Institute for Genetic Engineering and Biotechnology, Gebze Kocaeli 41470 (Turkey); Tuerker, Lemi [Department of Chemistry, Middle East Technical University, Ankara (Turkey); Eroglu, Inci [Department of Chemical Engineering, Middle East Technical University, Ankara (Turkey)

2006-09-15

In Rhodobacter capsulatus excess reducing equivalents generated by organic acid oxidation is consumed to reduce protons into hydrogen by the activity of nitrogenase. Nitrogenase serves as a redox-balancing tool and is activated by the RegB/RegA global regulatory system during photosynthetic growth. The terminal cytochrome cbb{sub 3} oxidase and the redox state of the cyclic photosynthetic electron transfer chain serve redox signaling to the RegB/RegA regulatory systems in Rhodobacter. In this study, hydrogen production of various R. capsulatus strains harboring the genetically modified electron carrier cytochromes or lacking the cyt cbb{sub 3} oxidase or the quinol oxidase were compared with the wild type. The results indicated that hydrogen production of mutant strains with modified electron carrier cytochromes decreased 3- to 4-fold, but the rate of hydrogen production increased significantly in a cbb{sub 3}{sup -} mutant. Moreover, hydrogen production efficiency of various R. capsulatus strains further increased by inactivation of uptake hydrogenase genes. (author)

Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced rhodobacter capsulatus cytochrome c(2) to the cytochrome bc(1) complex mediated by the conformation of the rieske iron-sulfur protein

International Nuclear Information System (INIS)

Devanathan, S.; Salamon, Z.; Tollin, G.; Fitch, J.C.; Meyer, T.E.; Berry, E.A.; Cusanovich, M.A.

2007-01-01

The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 M) but binds much more weakly to the oxidized form (Kd = 3.1 M). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 M. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 M) and reduced cytochrome c2 binds less strongly (Kd = 0.11 M) but ∼30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c

Resonance Raman and surface-enhanced resonance Raman spectra of LH2 antenna complex from Rhodobacter sphaeroides and Ectothiorhodospira sp. excited in the Qx and Qy transitions.

Science.gov (United States)

Chumanov, G; Picorel, R; Ortiz de Zarate, I; Cotton, T M; Seibert, M

2000-05-01

Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Qx and the Qy transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Qy region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment-pigment and pigment-protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment-protein complexes from different sources or in complexes altered chemically or genetically.

Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy.

Science.gov (United States)

Scheuring, Simon; Busselez, Johan; Lévy, Daniel

2005-01-14

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.

Dynamics of energy transfer from lycopene to bacteriochlorophyll in genetically-modified LH2 complexes of Rhodobacter sphaeroides.

Science.gov (United States)

Hörvin Billsten, H; Herek, J L; Garcia-Asua, G; Hashøj, L; Polívka, T; Hunter, C N; Sundström, V

2002-03-26

LH2 complexes from Rb. sphaeroides were modified genetically so that lycopene, with 11 saturated double bonds, replaced the native carotenoids which contain 10 saturated double bonds. Tuning the S1 level of the carotenoid in LH2 in this way affected the dynamics of energy transfer within LH2, which were investigated using both steady-state and time-resolved techniques. The S1 energy of lycopene in n-hexane was determined to be approximately 12 500 +/- 150 cm(-1), by direct measurement of the S1-S2 transient absorption spectrum using a femtosecond IR-probing technique, thus placing an upper limit on the S1 energy of lycopene in the LH2 complex. Fluorescence emission and excitation spectra demonstrated that energy can be transferred from lycopene to the bacteriochlorophyll molecules within this LH2 complex. The energy-transfer dynamics within the mutant complex were compared to wild-type LH2 from Rb. sphaeroides containing the carotenoid spheroidene and from Rs. molischianum, in which lycopene is the native carotenoid. The results show that the overall efficiency for Crt --> B850 energy transfer is approximately 80% in lyco-LH2 and approximately 95% in WT-LH2 of Rb. sphaeroides. The difference in overall Crt --> BChl transfer efficiency of lyco-LH2 and WT-LH2 mainly relates to the low efficiency of the Crt S(1) --> BChl pathway for complexes containing lycopene, which was 20% in lyco-LH2. These results show that in an LH2 complex where the Crt S1 energy is sufficiently high to provide efficient spectral overlap with both B800 and B850 Q(y) states, energy transfer via the Crt S1 state occurs to both pigments. However, the introduction of lycopene into the Rb. sphaeroides LH2 complex lowers the S1 level of the carotenoid sufficiently to prevent efficient transfer of energy to the B800 Q(y) state, leaving only the Crt S1 --> B850 channel, strongly suggesting that Crt S1 --> BChl energy transfer is controlled by the relative Crt S1 and BChl Q(y) energies.

The crystal structures of the tri-functional Chloroflexus aurantiacus and bi-functional Rhodobacter sphaeroides malyl-CoA lyases and comparison with CitE-like superfamily enzymes and malate synthases.

Science.gov (United States)

Zarzycki, Jan; Kerfeld, Cheryl A

2013-11-09

Malyl-CoA lyase (MCL) is a promiscuous carbon-carbon bond lyase that catalyzes the reversible cleavage of structurally related Coenzyme A (CoA) thioesters. This enzyme plays a crucial, multifunctional role in the 3-hydroxypropionate bi-cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus. A second, phylogenetically distinct MCL from Rhodobacter sphaeroides is involved in the ethylmalonyl-CoA pathway for acetate assimilation. Both MCLs belong to the large superfamily of CitE-like enzymes, which includes the name-giving β-subunit of citrate lyase (CitE), malyl-CoA thioesterases and other enzymes of unknown physiological function. The CitE-like enzyme superfamily also bears sequence and structural resemblance to the malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or both (the malate synthases). Here we report the first crystal structures of MCLs from two different phylogenetic subgroups in apo- and substrate-bound forms. Both the C. aurantiacus and the R. sphaeroides MCL contain elaborations on the canonical β8/α8 TIM barrel fold and form hexameric assemblies. Upon ligand binding, changes in the C-terminal domains of the MCLs result in closing of the active site, with the C-terminal domain of one monomer forming a lid over and contributing side chains to the active site of the adjacent monomer. The distinctive features of the two MCL subgroups were compared to known structures of other CitE-like superfamily enzymes and to malate synthases, providing insight into the structural subtleties that underlie the functional versatility of these enzymes. Although the C. aurantiacus and the R. sphaeroides MCLs have divergent primary structures (~37% identical), their tertiary and quaternary structures are very similar. It can be assumed that the C-C bond formation catalyzed by the MCLs occurs as proposed for

Monomeric RC-LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides.

Science.gov (United States)

Adams, Peter G; Mothersole, David J; Ng, Irene W; Olsen, John D; Hunter, C Neil

2011-09-01

In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre-light-harvesting 1-PufX (RC-LH1-PufX) 'core' complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX(-)). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX(-) mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC-LH1-PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX(-) membranes, resulting in locally ordered clusters of monomeric RC-LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation. 2011 Elsevier B.V. All rights reserved.

The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase.

Science.gov (United States)

Woronowicz, Kamil; Sha, Daniel; Frese, Raoul N; Sturgis, James N; Nanda, Vikas; Niederman, Robert A

2011-08-01

Atomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization.

Spectral diffusion and electron-phonon coupling of the B800 BChl a molecules in LH2 complexes from three different species of purple bacteria.

Science.gov (United States)

Baier, J; Gabrielsen, M; Oellerich, S; Michel, H; van Heel, M; Cogdell, R J; Köhler, J

2009-11-04

We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the alphabeta-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.

Photodynamics of BLUF domain proteins: a new class of the biological blue-light photoreceptors

OpenAIRE

Zirak Yousefabadi, Peyman

2008-01-01

BLUF domains are light sensors of many microorganisms. They are present in the multi-domain proteins e.g. AppA from the phototrophic proteobacterium Rhodobacter sphaeroides, YcgF from Escherichia coli, PAC (photoactive adenylyl cyclase) from the unicellular flagellate Euglena gracilis and single domain proteins e.g. BlrB from Rhodobacter sphaeroides, Slr1694 from cyanobacterium Synechocystis sp.PCC6803, and Tll0078 of the thermophilic unicellular cyanobacterium Thermosynechococcus elongates B...

Role of Rhodobacter sphaeroides photosynthetic reaction center residue M214 in the composition, absorbance properties, and conformations of H(A) and B(A) cofactors.

Science.gov (United States)

Saer, Rafael G; Hardjasa, Amelia; Rosell, Federico I; Mauk, A Grant; Murphy, Michael E P; Beatty, J Thomas

2013-04-02

In the native reaction center (RC) of Rhodobacter sphaeroides, the side chain of (M)L214 projects orthogonally toward the plane and into the center of the A branch bacteriopheophytin (BPhe) macrocycle. The possibility that this side chain is responsible for the dechelation of the central Mg(2+) of bacteriochlorophyll (BChl) was investigated by replacement of (M)214 with residues possessing small, nonpolar side chains that can neither coordinate nor block access to the central metal ion. The (M)L214 side chain was also replaced with Cys, Gln, and Asn to evaluate further the requirements for assembly of the RC with BChl in the HA pocket. Photoheterotrophic growth studies showed no difference in growth rates of the (M)214 nonpolar mutants at a low light intensity, but the growth of the amide-containing mutants was impaired. The absorbance spectra of purified RCs indicated that although absorbance changes are associated with the nonpolar mutations, the nonpolar mutant RC pigment compositions are the same as in the wild-type protein. Crystal structures of the (M)L214G, (M)L214A, and (M)L214N mutants were determined (determined to 2.2-2.85 Å resolution), confirming the presence of BPhe in the HA pocket and revealing alternative conformations of the phytyl tail of the accessory BChl in the BA site of these nonpolar mutants. Our results demonstrate that (i) BChl is converted to BPhe in a manner independent of the aliphatic side chain length of nonpolar residues replacing (M)214, (ii) BChl replaces BPhe if residue (M)214 has an amide-bearing side chain, (iii) (M)214 side chains containing sulfur are not sufficient to bind BChl in the HA pocket, and (iv) the (M)214 side chain influences the conformation of the phytyl tail of the BA BChl.

Bioaugmentation of Lactobacillus delbrueckii ssp. bulgaricus TISTR 895 to enhance bio-hydrogen production of Rhodobacter sphaeroides KKU-PS5.

Science.gov (United States)

Laocharoen, Sucheera; Reungsang, Alissara; Plangklang, Pensri

2015-01-01

Bioaugmentation or an addition of the desired microorganisms or specialized microbial strains into the anaerobic digesters can enhance the performance of microbial community in the hydrogen production process. Most of the studies focused on a bioaugmentation of native microorganisms capable of producing hydrogen with the dark-fermentative hydrogen producers while information on bioaugmentation of purple non-sulfur photosynthetic bacteria (PNSB) with lactic acid-producing bacteria (LAB) is still limited. In our study, bioaugmentation of Rhodobacter sphaeroides KKU-PS5 with Lactobacillus delbrueckii ssp. bulgaricus TISTR 895 was conducted as a method to produce hydrogen. Unfortunately, even though well-characterized microorganisms were used in the fermentation system, a cultivation of two different organisms in the same bioreactor was still difficult because of the differences in their metabolic types, optimal conditions, and nutritional requirements. Therefore, evaluation of the physical and chemical factors affecting hydrogen production of PNSB augmented with LAB was conducted using a full factorial design followed by response surface methodology (RSM) with central composite design (CCD). A suitable LAB/PNSB ratio and initial cell concentration were found to be 1/12 (w/w) and 0.15 g/L, respectively. The optimal initial pH, light intensity, and Mo concentration obtained from RSM with CCD were 7.92, 8.37 klux and 0.44 mg/L, respectively. Under these optimal conditions, a cumulative hydrogen production of 3396 ± 66 mL H2/L, a hydrogen production rate (HPR) of 9.1 ± 0.2 mL H2/L h, and a hydrogen yield (HY) of 9.65 ± 0.23 mol H2/mol glucose were obtained. KKU-PS5 augmented with TISTR 895 produced hydrogen from glucose at a relatively high HY, 9.65 ± 0.23 mol H2/mol glucose, i.e., 80 % of the theoretical yield. The ratio of the strains TISTR 895/KKU-PS5 and their initial cell concentrations affected the rate of lactic acid production and its

The unusually strong hydrogen bond between the carbonyl of Q(A) and His M219 in the Rhodobacter sphaeroides reaction center is not essential for efficient electron transfer from Q(A)(-) to Q(B).

Science.gov (United States)

Breton, Jacques; Lavergne, Jérôme; Wakeham, Marion C; Nabedryk, Eliane; Jones, Michael R

2007-06-05

In native reaction centers (RCs) from photosynthetic purple bacteria the primary quinone (QA) and the secondary quinone (QB) are interconnected via a specific His-Fe-His bridge. In Rhodobacter sphaeroides RCs the C4=O carbonyl of QA forms a very strong hydrogen bond with the protonated Npi of His M219, and the Ntau of this residue is in turn coordinated to the non-heme iron atom. The second carbonyl of QA is engaged in a much weaker hydrogen bond with the backbone N-H of Ala M260. In previous work, a Trp side chain was introduced by site-directed mutagenesis at the M260 position in the RC of Rb. sphaeroides, resulting in a complex that is completely devoid of QA and therefore nonfunctional. A photochemically competent derivative of the AM260W mutant was isolated that contains a Cys side chain at the M260 position (denoted AM260(W-->C)). In the present work, the interactions between the carbonyl groups of QA and the protein in the AM260(W-->C) suppressor mutant have been characterized by light-induced FTIR difference spectroscopy of the photoreduction of QA. The QA-/QA difference spectrum demonstrates that the strong interaction between the C4=O carbonyl of QA and His M219 is lost in the mutant, and the coupled CO and CC modes of the QA- semiquinone are also strongly perturbed. In parallel, a band assigned to the perturbation of the C5-Ntau mode of His M219 upon QA- formation in the native RC is lacking in the spectrum of the mutant. Furthermore, a positive band between 2900 and 2400 cm-1 that is related to protons fluctuating within a network of highly polarizable hydrogen bonds in the native RC is reduced in amplitude in the mutant. On the other hand, the QB-/QB FTIR difference spectrum is essentially the same as for the native RC. The kinetics of electron transfer from QA- to QB were measured by the flash-induced absorption changes at 780 nm. Compared to native RCs the absorption transients are slowed by a factor of about 2 for both the slow phase (in the

Continuous Cultivation of Photosynthetic Bacteria for Fatty Acids Production

DEFF Research Database (Denmark)

Kim, Dong-Hoon; Lee, Ji-Hye; Hwang, Yuhoon

2013-01-01

In the present work, we introduced a novel approach for microbial fatty acids (FA) production. Photosynthetic bacteria, Rhodobacter sphaeroides KD131, were cultivated in a continuous-flow, stirred-tank reactor (CFSTR) at various substrate (lactate) concentrations.At hydraulic retention time (HRT)....... sphaeroides was around 35% of dry cell weight, mainly composed of vaccenic acid (C18:1, omega-7)....

Membrane Curvature Induced by Aggregates of LH2s and Monomeric LH1s

OpenAIRE

Chandler, Danielle E.; Gumbart, James; Stack, John D.; Chipot, Christophe; Schulten, Klaus

2009-01-01

The photosynthetic apparatus of purple bacteria is contained within organelles called chromatophores, which form as extensions of the cytoplasmic membrane. The shape of these chromatophores can be spherical (as in Rhodobacter sphaeroides), lamellar (as in Rhodopseudomonas acidophila and Phaeospirillum molischianum), or tubular (as in certain Rb. sphaeroides mutants). Chromatophore shape is thought to be influenced by the integral membrane proteins Light Harvesting Complexes I and II (LH1 and ...

Imaging of solid tumor using near-infrared emitting purple bacteria

International Nuclear Information System (INIS)

Moon, Sung Min; Min, Jung Joon; Kim, Sun A; Choy, Hyon E.; Bom, Hee Seung

2005-01-01

Rhodobacter sphaeroides 2.4.1 is α-3 purple nonsulfur eubacterium with an extensive metabolism. Under anaerobic conditions, it is able to grow by photosynthesis, respiration and fermentation. When grown photosynthetically, it uses wavelengths of light in the near-infrared and contains a reaction center that is the peripheral light-harvesting (LH2) complex. These molecules absorb and emit near-infrared light. Using this near-infrared fluorescent bacterial we investigated its targeting capacity of solid tumor in small animals. R. sphaeroides 2.4.1 strains were cultured in sistrons minimal medium A (SIS) at 32 C. Xenograft tumor model has been established by subcutaneous injection of CT26 mouse colon cancer cell line. 1X10 8 Rhodobacter sphaeroides cells suspended in 100 ul of PBS were injected via tail vein with 1-cc insulin syringe into tumor bearing mouse. In vivo fluorescence imaging has been done after 20 min to 30 days of purple bacteria using indocyanine (ICG) emission filter (Em=810∼835 nm). Near-infrared imaging signal from Rhodobacter sphaeroides was initially detected at liver for 3 days but at the necrotic region of tumor mass thereafter. Total photon flux measured 5.5X10 8 (p/s/cm 2 /sr) at Day 1. Also it was increased to 7.8X10 8 (p/s/cm 2 /sr) at 12 day. One of important characteristic is that the signal appeared only at central necrosis area. It has been monitored for 36 day. We successfully imaged cancer with near-infrared fluorescence bacteria. Our result indicate that near-infrared fluorescence purple bacteria are able to be used to monitor bacterial trafficking in living tumor models

Paepalanthus sphaeroides, a new species of Eriocaulaceae from the Atlantic Forest, Brazil

NARCIS (Netherlands)

Trovó, M.; Echternacht, L.; Sano, P.T.

2012-01-01

We describe and illustrate Paepalanthus sphaeroides (Eriocaulaceae, Paepalanthoideae) from the Mantiqueira Range in South-eastern Brazil and compare it with the morphologically most similar species: Paepalanthus aequalis and Paepalanthus eriophaeus. Paepalanthus sphaeroides has unique membranaceous

NCBI nr-aa BLAST: CBRC-ATHA-01-0056 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-ATHA-01-0056 ref|YP_354669.1| ABC nitrate/sulfonate/bicarbonate transporter fa...mily, periplasmic substrate-binding protein [Rhodobacter sphaeroides 2.4.1] gb|ABA80768.1| ABC nitrate/sulfonate/bicarbonate

Biosynthesis of Chlorophyll a in a Purple Bacterial Phototroph and Assembly into a Plant Chlorophyll-Protein Complex.

Science.gov (United States)

Hitchcock, Andrew; Jackson, Philip J; Chidgey, Jack W; Dickman, Mark J; Hunter, C Neil; Canniffe, Daniel P

2016-09-16

Improvements to photosynthetic efficiency could be achieved by manipulating pigment biosynthetic pathways of photosynthetic organisms in order to increase the spectral coverage for light absorption. The development of organisms that can produce both bacteriochlorophylls and chlorophylls is one way to achieve this aim, and accordingly we have engineered the bacteriochlorophyll-utilizing anoxygenic phototroph Rhodobacter sphaeroides to make chlorophyll a. Bacteriochlorophyll and chlorophyll share a common biosynthetic pathway up to the precursor chlorophyllide. Deletion of genes responsible for the bacteriochlorophyll-specific modifications of chlorophyllide and replacement of the native bacteriochlorophyll synthase with a cyanobacterial chlorophyll synthase resulted in the production of chlorophyll a. This pigment could be assembled in vivo into the plant water-soluble chlorophyll protein, heterologously produced in Rhodobacter sphaeroides, which represents a proof-of-principle for the engineering of novel antenna complexes that enhance the spectral range of photosynthesis.

Investigation of the effects of different carotenoids on the absorption and CD signals of light harvesting 1 complexes

NARCIS (Netherlands)

Georgakopoulou, S.; van der Zwan, G.; Olsen, J.D.; Hunter, C.N.; Niederman, R.A.; van Grondelle, R.

2006-01-01

Absorption and circular dichroism (CD) spectra of light-harvesting (LH)1 complexes from the purple bacteria Rhodobacter (Rba.) sphaeroides and Rhodospirillum (Rsp.) rubrum are presented. The complexes exhibit very low intensity, highly nonconservative, near-infrared (NIR) CD spectra. Absorption and

Respiratory Syncytial Virus Fusion Protein-Induced Toll-Like Receptor 4 (TLR4) Signaling Is Inhibited by the TLR4 Antagonists Rhodobacter sphaeroides Lipopolysaccharide and Eritoran (E5564) and Requires Direct Interaction with MD-2

Science.gov (United States)

Rallabhandi, Prasad; Phillips, Rachel L.; Boukhvalova, Marina S.; Pletneva, Lioubov M.; Shirey, Kari Ann; Gioannini, Theresa L.; Weiss, Jerrold P.; Chow, Jesse C.; Hawkins, Lynn D.; Vogel, Stefanie N.; Blanco, Jorge C. G.

2012-01-01

ABSTRACT Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2−/− macrophages, where MD-2−/− macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4–CD14–MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology. PMID:22872782

Long-Range Energy Propagation in Nanometer Arrays of Light Harvesting Antenna Complexes

NARCIS (Netherlands)

Escalantet, Maryana; Escalante Marun, M.; Lenferink, Aufrid T.M.; Zhao, Yiping; Tas, Niels Roelof; Huskens, Jurriaan; Hunter, C. Neil; Subramaniam, Vinod; Otto, Cornelis

2010-01-01

Here we report the first observation of long-range transport of excitation energy within a biomimetic molecular nanoarray constructed from LH2 antenna complexes from Rhodobacter sphaeroides. Fluorescence microscopy of the emission of light after local excitation with a diffraction-limited light beam

Bacterial PerO Permeases Transport Sulfate and Related Oxyanions.

Science.gov (United States)

Hoffmann, Marie-Christine; Pfänder, Yvonne; Tintel, Marc; Masepohl, Bernd

2017-07-15

Rhodobacter capsulatus synthesizes the high-affinity ABC transporters CysTWA and ModABC to specifically import the chemically related oxyanions sulfate and molybdate, respectively. In addition, R. capsulatus has the low-affinity permease PerO acting as a general oxyanion transporter, whose elimination increases tolerance to molybdate and tungstate. Although PerO-like permeases are widespread in bacteria, their function has not been examined in any other species to date. Here, we present evidence that PerO permeases from the alphaproteobacteria Agrobacterium tumefaciens , Dinoroseobacter shibae , Rhodobacter sphaeroides , and Sinorhizobium meliloti and the gammaproteobacterium Pseudomonas stutzeri functionally substitute for R. capsulatus PerO in sulfate uptake and sulfate-dependent growth, as shown by assimilation of radioactively labeled sulfate and heterologous complementation. Disruption of perO genes in A. tumefaciens , R. sphaeroides , and S. meliloti increased tolerance to tungstate and, in the case of R. sphaeroides , to molybdate, suggesting that heterometal oxyanions are common substrates of PerO permeases. This study supports the view that bacterial PerO permeases typically transport sulfate and related oxyanions and, hence, form a functionally conserved permease family. IMPORTANCE Despite the widespread distribution of PerO-like permeases in bacteria, our knowledge about PerO function until now was limited to one species, Rhodobacter capsulatus In this study, we showed that PerO proteins from diverse bacteria are functionally similar to the R. capsulatus prototype, suggesting that PerO permeases form a conserved family whose members transport sulfate and related oxyanions. Copyright © 2017 American Society for Microbiology.

Role of charge-transfer states in bacterial photosynthesis

NARCIS (Netherlands)

Meech, S.R.; Hoff, A.J.

1986-01-01

Photon echo, photon-echo excitation, and "hole-burning" data recorded in the 800-990 nm region of Rhodobacter sphaeroides R26 and Rhodopseudomonas viridis reaction centers are reported. The primary process in these reaction centers, following excitation, was found to occur in ≈25 fsec; the

Molecular Regulation of Photosynthetic Carbon Dioxide Fixation in Nonsulfur Purple Bacteria

Energy Technology Data Exchange (ETDEWEB)

Tabita, Fred Robert [The Ohio State Univ., Columbus, OH (United States)

2015-12-01

The overall objective of this project is to determine the mechanism by which a transcriptional activator protein affects CO₂ fixation (cbb) gene expression in nonsulfur purple photosynthetic bacteria, with special emphasis to Rhodobacter sphaeroides and with comparison to Rhodopseudomonas palustris. These studies culminated in several publications which indicated that additional regulators interact with the master regulator CbbR in both R. sphaeroides and R. palustris. In addition, the interactive control of the carbon and nitrogen assimilatory pathways was studied and unique regulatory signals were discovered.

Antagonistic Activities of Purple Non-sulfur Bacterial Extracts Against Antibiotic Resistant Vibrio sp.

Directory of Open Access Journals (Sweden)

Chandrasekaran, R.

2011-01-01

Full Text Available Solvent extracts of native purple non-sulfur bacterial (PNSB isolates from the effluents of brackish shrimp culture ponds, near Nagapattinam coast (South India were evaluated for antibacterial activity by the disc diffusion method. Best results were shown by the chloroform extracts against oxytetracycline resistant Vibrio harveyi and Vibrio fischerii. Among the purple non-sulfur bacterial isolates, Rhodobacter sphaeroides, showed maximum antagonistic activity. The findings suggest that the antagonistic extracts from Rba. sphaeroides could be used as an effective antibiotic in controlling Vibrio spp., in aquaculture systems.

Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers

NARCIS (Netherlands)

Visschers, R.W.; Vulto, S.I.E.; Jones, M.R.; van Grondelle, R.; Kraayenhof, R.

1999-01-01

The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for

Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers.

NARCIS (Netherlands)

Visschers, R.W.; Vulto, S.I.E.; Jones, M.R.; van Grondelle, R.; Kraayenhof, R.

1999-01-01

The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for

Photocycle of the flavin-binding photoreceptor AppA, a bacterial transcriptional antirepressor of photosynthesis genes

NARCIS (Netherlands)

Gauden, M.L.; Yeremenko, S.; Laan, W.; van Stokkum, I.H.M.; Ihalainen, J.A.; van Grondelle, R.; Hellingwerf, K.J.; Kennis, J.T.M.

2005-01-01

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal domain belonging to a new class of photoreceptors designated BLUF domains. AppA was shown to control photosynthesis gene expression in response to blue light and oxygen tension. We have investigated the photocycle of the AppA

GenBank blastx search result: AK061856 [KOME

Lifescience Database Archive (English)

Full Text Available AK061856 001-040-G03 AF018073.1 Rhodobacter sphaeroides operon regulator (smoC), periplasmic sorbitol...-binding protein (smoE), sorbitol/mannitol transport inner membrane protein (smoF), sorbitol.../mannitol transport inner membrane protein (smoG), sorbitol/mannitol transport ATP-binding transport protein (smoK), sorbitol

Conserved Chloroplast Open-reading Frame ycf54 Is Required for Activity of the Magnesium Protoporphyrin Monomethylester Oxidative Cyclase in Synechocystis PCC 6803

Czech Academy of Sciences Publication Activity Database

Hollingshead, S.; Kopečná, Jana; Jackson, P. J.; Canniffe, D. P.; Davidson, P. A.; Dickman, M. J.; Sobotka, Roman; Hunter, C. N.

2012-01-01

Roč. 287, č. 33 (2012), s. 27823-27833 ISSN 0021-9258 R&D Projects: GA ČR GAP501/10/1000; GA MŠk(CZ) ED2.1.00/03.0110 Institutional support: RVO:61388971 Keywords : CHLOROPHYLL ISOCYCLIC RING * RHODOBACTER-SPHAEROIDES * SP PCC-6803 Subject RIV: CE - Biochemistry Impact factor: 4.651, year: 2012

Effects of light intensity and quality on phycobiliprotein accumulation in the cyanobacterium Nostoc sphaeroides Kützing.

Science.gov (United States)

Ma, Rui; Lu, Fan; Bi, Yonghong; Hu, Zhengyu

2015-08-01

To assess the effects of light intensity and quality on the growth and phycobiliproteins (PBP) accumulation in Nostoc sphaeroides Kützing (N. sphaeroides). Dry weights, dry matter, protein, chlorophyll and PBP contents were higher under 90 μmol m(-2) s(-1) than under other intensities (both higher and lower). Phycocyanin and allophycocyanin increased with light intensity while phycoerythrin decreased. Fresh weights, protein and PBP contents increased at the highest rates under blue light. Red light resulted in higher values of dry matter, phycocyanin and chlorophyll a. White light at 90 μmol m(-2) s(-1) or blue light 30 μmol m(-2) s(-1) were optimal for the growth and phycobiliprotein accumulation in N. sphaeroides.

Electrostatic dominoes: long distance propagation of mutational effects in photosynthetic reaction centers of Rhodobacter capsulatus.

Science.gov (United States)

Sebban, P; Maróti, P; Schiffer, M; Hanson, D K

1995-07-04

Two point mutants from the purple bacterium Rhodobacter capsulatus, both modified in the M protein of the photosynthetic reaction center, have been studied by flash-induced absorbance spectroscopy. These strains carry either the M231Arg --> Leu or M43ASN --> Asp mutations, which are located 9 and 15 A, respectively, from the terminal electron acceptor QB. In the wild-type Rb. sphaeroides structure, M231Arg is involved in a conserved salt bridge with H125Glu and H232Glu and M43Asn is located among several polar residues that form or surround the QB binding site. These substitutions were originally uncovered in phenotypic revertants isolated from the photosynthetically incompetent L212Glu-L213Asp --> Ala-Ala site-specific double mutant. As second-site suppressor mutations, they have been shown to restore the proton transfer function that is interrupted in the L212Ala-L213Ala double mutant. The electrostatic effects that are induced in reaction centers by the M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the same in either the double-mutant or wild-type backgrounds. In a reaction center that is otherwise wild type in sequence, they decrease the free energy gap between the QA- and QB- states by 24 +/- 5 and 45 +/- 5 meV, respectively. The pH dependences of K2, the QA-QB QAQB- equilibrium constant, are altered in reaction centers that carry either of these substitutions, revealing differences in the pKas of titratable groups compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Nostoc sphaeroides Kützing, an excellent candidate producer for CELSS

Science.gov (United States)

Hao, Zongjie; Li, Dunhai; Li, Yanhui; Wang, Zhicong; Xiao, Yuan; Wang, Gaohong; Liu, Yongding; Hu, Chunxiang; Liu, Qifang

2011-11-01

Some phytoplankton can be regarded as possible candidates in the establishment of Controlled Ecological Life Support System (CELSS) for some intrinsic characteristics, the first characteristic is that they should grow rapidly, secondly, they should be able to endure some stress factors and develop some corresponding adaptive strategies; also it is very important that they could provide food rich in nutritious protein and vitamins for the crew; the last but not the least is they can also fulfill the other main functions of CELSS, including supplying oxygen, removing carbon dioxide and recycling the metabolic waste. According to these characteristics, Nostoc sphaeroides, a potential healthy food in China, was selected as the potential producer in CELSS. It was found that the oxygen average evolution rate of this algae is about 150 μmol O 2 mg -1 h -1, and the size of them are ranged from 2 to 20 mm. Also it can be cultured with high population density, which indicated that the potential productivity of Nostoc sphaeroides is higher than other algae in limited volume. We measured the nutrient contents of the cyanobacterium and concluded it was a good food for the crew. Based on above advantages, Nostoc sphaeroides was assumed to a suitable phytoplankton for the establishment of Controlled Ecological Life Support System. We plan to develop suitable bioreactor with the cyanobacterium for supplying oxygen and food in future space missions.

GenBank blastx search result: AK241729 [KOME

Lifescience Database Archive (English)

Full Text Available AK241729 J065199L10 AF018073.1 AF018073 Rhodobacter sphaeroides operon regulator (smoC), periplasmic sorbito...l-binding protein (smoE), sorbitol/mannitol transport inner membrane protein (smoF), sorbitol.../mannitol transport inner membrane protein (smoG), sorbitol/mannitol transport ATP-binding transport protein (smoK), sorbit...ol dehydrogenase (smoS), mannitol dehydrogenase (mtlK),

GenBank blastx search result: AK240874 [KOME

Lifescience Database Archive (English)

Full Text Available AK240874 J065025K09 AF018073.1 AF018073 Rhodobacter sphaeroides operon regulator (smoC), periplasmic sorbito...l-binding protein (smoE), sorbitol/mannitol transport inner membrane protein (smoF), sorbitol.../mannitol transport inner membrane protein (smoG), sorbitol/mannitol transport ATP-binding transport protein (smoK), sorbit...ol dehydrogenase (smoS), mannitol dehydrogenase (mtlK),

GenBank blastx search result: AK243680 [KOME

Lifescience Database Archive (English)

Full Text Available AK243680 J100090I20 AF018073.1 AF018073 Rhodobacter sphaeroides operon regulator (smoC), periplasmic sorbito...l-binding protein (smoE), sorbitol/mannitol transport inner membrane protein (smoF), sorbitol.../mannitol transport inner membrane protein (smoG), sorbitol/mannitol transport ATP-binding transport protein (smoK), sorbit...ol dehydrogenase (smoS), mannitol dehydrogenase (mtlK),

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

Science.gov (United States)

Gray, K A; Dutton, P L; Daldal, F

1994-01-25

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

Effects of radio frequency and high pressure steam sterilisation on the colour and flavour of prepared Nostoc sphaeroides.

Science.gov (United States)

Xu, Jicheng; Zhang, Min; An, Yanjun; Roknul, Azam Sm; Adhikari, Benu

2018-03-01

Nostoc sphaeroides has been used as a highly effective herbal medicine and dietary supplement for thousands of years. The desired dark green colour of fresh N. sphaeroides is converted into an undesirable dark brown during conventional high pressure (HP) steam sterilisation. Radio frequency (RF) sterilisation technology was used in this study to determine its effectiveness in sterilising N. sphaeroides and to achieve better preservation of natural colour and desirable flavour. Sterilisation was carried out using a 6 kW, 27 MHz RF instrument for 10, 20 and 30 min. The degree of microbial kill and the effects of RF sterilisation on colour and flavour were determined and compared with those obtained from HP steam (121 °C, 30 min) sterilisation. The effects of RF sterilisation on colour and flavour (measured using electronic nose) parameters were significantly lower than that in HP steam sterilisation. The RF sterilisation carried out for 20 min achieved logarithmic reduction of bacterial population and met China's national standard while preserving the colour and flavour better. Results of the present study indicated that application of RF sterilisation would improve the quality of sterilised N. sphaeroides and broaden its application in the food and health food industries. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Conformational regulation of charge recombination reactions in a photosynthetic bacterial reaction center

DEFF Research Database (Denmark)

Katona, Gergely; Snijder, Arjan; Gourdon, Pontus Emanuel

2005-01-01

In bright light the photosynthetic reaction center (RC) of Rhodobacter sphaeroides stabilizes the P(+)(870).Q(-)(A) charge-separated state and thereby minimizes the potentially harmful effects of light saturation. Using X-ray diffraction we report a conformational change that occurs within the cy...... the cytoplasmic domain of this RC in response to prolonged illumination with bright light. Our observations suggest a novel structural mechanism for the regulation of electron transfer reactions in photosynthesis....

Overall energy conversion efficiency of a photosynthetic vesicle

Energy Technology Data Exchange (ETDEWEB)

Sener, Melih [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States; Strumpfer, Johan [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States; Singharoy, Abhishek [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Hunter, C. Neil [Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom; Schulten, Klaus [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States

2016-08-26

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

OpenAIRE

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-01-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of ...

Engineered photoproteins that give rise to photosynthetically-incompetent bacteria are effective as photovoltaic materials for biohybrid photoelectrochemical cells.

Science.gov (United States)

Liu, Juntai; Friebe, Vincent M; Swainsbury, David J K; Crouch, Lucy I; Szabo, David A; Frese, Raoul N; Jones, Michael R

2018-04-17

Reaction centre/light harvesting proteins such as the RCLH1X complex from Rhodobacter sphaeroides carry out highly quantum-efficient conversion of solar energy through ultrafast energy transfer and charge separation, and these pigment-proteins have been incorporated into biohybrid photoelectrochemical cells for a variety of applications. In this work we demonstrate that, despite not being able to support normal photosynthetic growth of Rhodobacter sphaeroides, an engineered variant of this RCLH1X complex lacking the PufX protein and with an enlarged light harvesting antenna is unimpaired in its capacity for photocurrent generation in two types of bio-photoelectrochemical cells. Removal of PufX also did not impair the ability of the RCLH1 complex to act as an acceptor of energy from synthetic light harvesting quantum dots. Unexpectedly, the removal of PufX led to a marked improvement in the overall stability of the RCLH1 complex under heat stress. We conclude that PufX-deficient RCLH1 complexes are fully functional in solar energy conversion in a device setting and that their enhanced structural stability could make them a preferred choice over their native PufX-containing counterpart. Our findings on the competence of RCLH1 complexes for light energy conversion in vitro are discussed with reference to the reason why these PufX-deficient proteins are not capable of light energy conversion in vivo.

The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase.

Science.gov (United States)

Bickar, D; Turrens, J F; Lehninger, A L

1986-11-05

When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two-electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c.

Reverse micelles as suitable microreactor for increased biohydrogen production

Energy Technology Data Exchange (ETDEWEB)

Pandey, Anjana [Nanotechnology and Molecular Biology Laboratory, Centre of Biotechnology, University of Allahabad, Allahabad 211002 (India); Pandey, Ashutosh [Centre of Energy Studies, MNNIT, Allahabad 211004 (India)

2008-01-15

Reverse micelles have been shown to act as efficient microreactors for enzymic reactions and whole cell entrapment in organic (non-aqueous) media wherein the reactants are protected from denaturation by the surrounding organic solvent. These micelles are thermodynamically stable, micrometer sized water droplets dispersed in an organic phase by a surfactant. It has been observed that when whole cells of photosynthetic bacteria (Rhodopseudomonas sphaeroides or Rhodobacter sphaeroides 2.4.1) are entrapped inside these reverse micelles, the H{sub 2} production enhanced from 25 to 35 folds. That is, 1.71mmol(mgprotein){sup -1}h{sup -1} in case of R. sphaeroides which is 25 fold higher in benzene-sodium lauryl sulfate reverse micelles. Whereas, in case of R. sphaeroides 2.4.1 the H{sub 2} production was increased by 35 fold within AOT-isooctane reverse micelles i.e. 11.5mmol(mgprotein){sup -1}h{sup -1}. The observations indicate that the entrapment of whole cells of microbes within reverse micelles provides a novel and efficient technique to produce hydrogen by the inexhaustible biological route. The two microorganisms R. sphaeroides 2.4.1 (a photosynthetic bacteria) and Citrobacter Y19 (a facultative anaerobic bacteria) together are also entrapped within AOT-isooctane and H{sub 2} production was measured i.e. 69mmol(mgprotein){sup -1}h{sup -1}. The nitrogenase enzyme responsible for hydrogen production by R. sphaeroides/R. sphaeroides 2.4.1 cells is oxygen sensitive, and very well protected within reverse micelles by the use of combined approach of two cells (R. sphaeroides 2.4.1 and Citrobacter Y19). In this case glucose present in the medium of Citrobacter Y19 serves double roles in enhancing the sustained production rate of hydrogen. Firstly, it quenches the free O{sub 2}liberated as a side product of reaction catalyzed by nitrogenase, which is O{sub 2} labile. Secondly, organic acid produced by this reaction is utilized by the Citrobacter Y19 as organic substrate in

Supplementing in the diet of lactating Holstein cows may naturally produce coenzyme Q10-enriched milk

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Gui-Seck Bae

2018-01-01

Full Text Available Objective To examine the effects of Rhodobacter sphaeroides (R. sphaeroides supplementation as a direct-fed microbial (DFM on rumen fermentation in dairy cows and on coenzyme Q10 (CoQ10 transition into milk, an in vitro rumen simulation batch culture and an in vivo dairy cow experiment were conducted. Methods The characteristics of in vitro ruminal fermentation were investigated using rumen fluids from six cannulated Holstein dairy cows at 2 h post-afternoon feeding. A control treatment was included in the experiments based on a typified total mixed ration (TMR for lactating dairy cows, which was identical to the one used in the in vivo study, plus R. sphaeroides at 0.1%, 0.3%, and 0.5% TMR dry matter. The in vivo study employed six ruminally cannulated lactating Holstein cows randomly allotted to either the control TMR (C-TMR treatment or to a diet supplemented with a 0.5% R. sphaeroides culture (S-TMR, dry matter basis ad libitum. The presence of R. sphaeroides was verified using denaturing gradient gel electrophoresis (DGGE applied to the bacterial samples obtained from the in vivo study. The concentration of CoQ10 in milk and in the supernatant from the in vitro study was determined using high performance liquid chromatography. Results The results of the in vitro batch culture and DGGE showed that the concentration of CoQ10 significantly increased after 2 h of R. sphaeroides supplementation above 0.1%. When supplemented to the diet of lactating cows at the level of 0.5%, R. sphaeroides did not present any adverse effect on dry matter intake and milk yield. However, the concentration of CoQ10 in milk dramatically increased, with treated cows producing 70.9% more CoQ10 than control cows. Conclusion The CoQ10 concentration in milk increased via the use of a novel DFM, and R. sphaeroides might be used for producing value-added milk and dairy products in the future.

Electron spin relaxation enhancement measurements of interspin distances in human, porcine, and Rhodobacter electron transfer flavoprotein ubiquinone oxidoreductase (ETF QO)

Science.gov (United States)

Fielding, Alistair J.; Usselman, Robert J.; Watmough, Nicholas; Simkovic, Martin; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

2008-02-01

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a membrane-bound electron transfer protein that links primary flavoprotein dehydrogenases with the main respiratory chain. Human, porcine, and Rhodobacter sphaeroides ETF-QO each contain a single [4Fe-4S] 2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated enzyme and become paramagnetic on reduction with the enzymatic electron donor or with dithionite. The anionic flavin semiquinone can be reduced further to diamagnetic hydroquinone. The redox potentials for the three redox couples are so similar that it is not possible to poise the proteins in a state where both the [4Fe-4S] + cluster and the flavoquinone are fully in the paramagnetic form. Inversion recovery was used to measure the electron spin-lattice relaxation rates for the [4Fe-4S] + between 8 and 18 K and for semiquinone between 25 and 65 K. At higher temperatures the spin-lattice relaxation rates for the [4Fe-4S] + were calculated from the temperature-dependent contributions to the continuous wave linewidths. Although mixtures of the redox states are present, it was possible to analyze the enhancement of the electron spin relaxation of the FAD semiquinone signal due to dipolar interaction with the more rapidly relaxing [4Fe-4S] + and obtain point-dipole interspin distances of 18.6 ± 1 Å for the three proteins. The point-dipole distances are within experimental uncertainty of the value calculated based on the crystal structure of porcine ETF-QO when spin delocalization is taken into account. The results demonstrate that electron spin relaxation enhancement can be used to measure distances in redox poised proteins even when several redox states are present.

Electron spin relaxation enhancement measurements of interspin distances in human, porcine, and Rhodobacter electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO).

Science.gov (United States)

Fielding, Alistair J; Usselman, Robert J; Watmough, Nicholas; Simkovic, Martin; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

2008-02-01

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a membrane-bound electron transfer protein that links primary flavoprotein dehydrogenases with the main respiratory chain. Human, porcine, and Rhodobacter sphaeroides ETF-QO each contain a single [4Fe-4S](2+,1+) cluster and one equivalent of FAD, which are diamagnetic in the isolated enzyme and become paramagnetic on reduction with the enzymatic electron donor or with dithionite. The anionic flavin semiquinone can be reduced further to diamagnetic hydroquinone. The redox potentials for the three redox couples are so similar that it is not possible to poise the proteins in a state where both the [4Fe-4S](+) cluster and the flavoquinone are fully in the paramagnetic form. Inversion recovery was used to measure the electron spin-lattice relaxation rates for the [4Fe-4S](+) between 8 and 18K and for semiquinone between 25 and 65K. At higher temperatures the spin-lattice relaxation rates for the [4Fe-4S](+) were calculated from the temperature-dependent contributions to the continuous wave linewidths. Although mixtures of the redox states are present, it was possible to analyze the enhancement of the electron spin relaxation of the FAD semiquinone signal due to dipolar interaction with the more rapidly relaxing [4Fe-4S](+) and obtain point-dipole interspin distances of 18.6+/-1A for the three proteins. The point-dipole distances are within experimental uncertainty of the value calculated based on the crystal structure of porcine ETF-QO when spin delocalization is taken into account. The results demonstrate that electron spin relaxation enhancement can be used to measure distances in redox poised proteins even when several redox states are present.

Nostoc sphaeroides Kütz, a candidate producer par excellence for CELSS

Science.gov (United States)

Wang, Gaohong; Hao, Zongjie; Liu, Yongding

A lot of aquatic organisms could be regarded as suitable candidates par excellence in the establishment of CELSS, since they are relatively easy and fast to grow and resistant to changes in environmental condition as well as providing nutritious, protein-and vitamin-rich foods for the crew, which can fulfill the main functions of CELSS, including supplying oxygen, water and food, removing carbon dioxide and making daily life waste reusable. Our labotory has developed mass culture of Nostoc sphaeroides Kütz, which is one of traditional healthy food in China and. The oxygen evolution rate of the cyanobacterium is about 150 molO2.mg-1.h-1, and it usually grows into colony with size between 2-20mm, which is easy to be harvested. It also can be cultured with high density, which show that the productivity of the cyanobacterium in limited volume is higher than other microalgae. We had measured the nutrient content of the cyanobacterium and developed some Chinese Dishes and Soups with Nostoc sphaeroides Kütz, which showed that it was a good food for crew. Using remote sensing technique, we also investigated its growth in Closed System under microgravity by SHENZHOU-2 spacecraft in January 2001. We plan to develop suitable bioreactor with the cyanobacterium for supplying oxygen and food to crew in future.

A complex of cardiac cytochrome c1 and cytochrome c.

Science.gov (United States)

Chiang, Y L; Kaminsky, L S; King, T E

1976-01-10

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of

The three-dimensional structures of bacterial reaction centers.

Science.gov (United States)

Olson, T L; Williams, J C; Allen, J P

2014-05-01

This review presents a broad overview of the research that enabled the structure determination of the bacterial reaction centers from Blastochloris viridis and Rhodobacter sphaeroides, with a focus on the contributions from Duysens, Clayton, and Feher. Early experiments performed in the laboratory of Duysens and others demonstrated the utility of spectroscopic techniques and the presence of photosynthetic complexes in both oxygenic and anoxygenic photosynthesis. The laboratories of Clayton and Feher led efforts to isolate and characterize the bacterial reaction centers. The availability of well-characterized preparations of pure and stable reaction centers allowed the crystallization and subsequent determination of the structures using X-ray diffraction. The three-dimensional structures of reaction centers revealed an overall arrangement of two symmetrical branches of cofactors surrounded by transmembrane helices from the L and M subunits, which also are related by the same twofold symmetry axis. The structure has served as a framework to address several issues concerning bacterial photosynthesis, including the directionality of electron transfer, the properties of the reaction center-cytochrome c 2 complex, and the coupling of proton and electron transfer. Together, these research efforts laid the foundation for ongoing efforts to address an outstanding question in oxygenic photosynthesis, namely the molecular mechanism of water oxidation.

Embryonic, Larval, and Early Juvenile Development of the Tropical Sea Urchin, Salmacis sphaeroides (Echinodermata: Echinoidea

Directory of Open Access Journals (Sweden)

M. Aminur Rahman

2012-01-01

Full Text Available Salmacis sphaeroides (Linnaeus, 1758 is one of the regular echinoids, occuring in the warm Indo-West Pacific, including Johor Straits, between Malaysia and Singapore. In order to investigate the developmental basis of morphological changes in embryos and larvae, we documented the ontogeny of S. sphaeroides in laboratory condition. Gametes were obtained from adult individuals by 0.5 M KCl injection into the coelomic cavity. Fertilization rate at limited sperm concentration (10−5 dilution was 96.6±1.4% and the resulting embryos were reared at 24°C. First cleavage (2-cell, 4-cell, 8-cell, 16-cell, 32-cell, and multicell (Morulla stages were achieved 01.12, 02.03, 02.28, 02.51, 03.12, and 03.32 h postfertilization. Ciliated blastulae with a mean length of 174.72±4.43 μm hatched 08.45 h after sperm entry. The gastrulae formed 16.15 h postfertilization and the archenteron elongated constantly while ectodermal red-pigmented cells migrated synchronously to the apical plate. Pluteus larva started to feed unicellular algae in 2 d, grew continuously, and finally attained metamorphic competence in 35 d after fertilization. Metamorphosis took approximately 1 h 30 min from attachment to the complete resorption of larval tissues and the development of complete juvenile structure with adult spines, extended tubefeet and well-developed pedicellaria, the whole event of which usually took place within 1 d postsettlement. This study represents the first successful investigation on embryonic, larval, and early juvenile development of S. sphaeroides. The findings would greatly be helpful towards the understanding of ontogeny and life-history strategies, which will facilitate us to develop the breeding, seed production, and culture techniques of sea urchins in captive condition.

Structural and phylogenetic analysis of Rhodobacter capsulatus NifF: uncovering general features of nitrogen-fixation (nif)-flavodoxins.

Science.gov (United States)

Pérez-Dorado, Inmaculada; Bortolotti, Ana; Cortez, Néstor; Hermoso, Juan A

2013-01-09

Analysis of the crystal structure of NifF from Rhodobacter capsulatus and its homologues reported so far reflects the existence of unique structural features in nif flavodoxins: a leucine at the re face of the isoalloxazine, an eight-residue insertion at the C-terminus of the 50's loop and a remarkable difference in the electrostatic potential surface with respect to non-nif flavodoxins. A phylogenetic study on 64 sequences from 52 bacterial species revealed four clusters, including different functional prototypes, correlating the previously defined as "short-chain" with the firmicutes flavodoxins and the "long-chain" with gram-negative species. The comparison of Rhodobacter NifF structure with other bacterial flavodoxin prototypes discloses the concurrence of specific features of these functional electron donors to nitrogenase.

Structural and Phylogenetic Analysis of Rhodobacter capsulatus NifF: Uncovering General Features of Nitrogen-fixation (nif-Flavodoxins

Directory of Open Access Journals (Sweden)

Inmaculada Pérez-Dorado

2013-01-01

Full Text Available Analysis of the crystal structure of NifF from Rhodobacter capsulatus and its homologues reported so far reflects the existence of unique structural features in nif flavodoxins: a leucine at the re face of the isoalloxazine, an eight-residue insertion at the C-terminus of the 50’s loop and a remarkable difference in the electrostatic potential surface with respect to non-nif flavodoxins. A phylogenetic study on 64 sequences from 52 bacterial species revealed four clusters, including different functional prototypes, correlating the previously defined as “short-chain” with the firmicutes flavodoxins and the “long-chain” with gram-negative species. The comparison of Rhodobacter NifF structure with other bacterial flavodoxin prototypes discloses the concurrence of specific features of these functional electron donors to nitrogenase.

Directed assembly of functional light harvesting antenna complexes onto chemically patterned surfaces

International Nuclear Information System (INIS)

Escalante, Maryana; Maury, Pascale; Bruinink, Christiaan M; Werf, Kees van der; Olsen, John D; Timney, John A; Huskens, Jurriaan; Hunter, C Neil; Subramaniam, Vinod; Otto, Cees

2008-01-01

We report the directed assembly of the photosynthetic membrane proteins LH1 and LH2 isolated from the purple bacterium Rhodobacter sphaeroides onto chemically patterned substrates. Nanoimprint lithography was used to pattern discrete regions of amino- and fluoro-terminated or poly(ethylene glycol) self-assembled monolayers onto a glass substrate. Densely packed layers of assembled protein complexes were observed with atomic force microscopy. The protein complexes attached selectively to the amino-terminated regions by electrostatic interactions. Spectral images generated with a hybrid scanning probe and fluorescence microscope confirmed that the patterned proteins retained their native optical signatures

Directed assembly of functional light harvesting antenna complexes onto chemically patterned surfaces

Energy Technology Data Exchange (ETDEWEB)

Escalante, Maryana [Biophysical Engineering Group, MESA and Institute for Nanotechnology, University of Twente, PO Box 217, 7500 AE Enschede (Netherlands); Maury, Pascale [Molecular Nanofabrication Group, MESA and Institute for Nanotechnology, University of Twente, PO Box 217, 7500 AE Enschede (Netherlands); Bruinink, Christiaan M [Molecular Nanofabrication Group, MESA and Institute for Nanotechnology, University of Twente, PO Box 217, 7500 AE Enschede (Netherlands); Werf, Kees van der [Biophysical Engineering Group, MESA and Institute for Nanotechnology, University of Twente, PO Box 217, 7500 AE Enschede (Netherlands); Olsen, John D [Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN (United Kingdom); Timney, John A [Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN (United Kingdom); Huskens, Jurriaan [Molecular Nanofabrication Group, MESA and Institute for Nanotechnology, University of Twente, PO Box 217, 7500 AE Enschede (Netherlands); Hunter, C Neil [Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN (United Kingdom); Subramaniam, Vinod [Biophysical Engineering Group, MESA and Institute for Nanotechnology, University of Twente, PO Box 217, 7500 AE Enschede (Netherlands); Otto, Cees [Biophysical Engineering Group, MESA and Institute for Nanotechnology, University of Twente, PO Box 217, 7500 AE Enschede (Netherlands)

2008-01-16

We report the directed assembly of the photosynthetic membrane proteins LH1 and LH2 isolated from the purple bacterium Rhodobacter sphaeroides onto chemically patterned substrates. Nanoimprint lithography was used to pattern discrete regions of amino- and fluoro-terminated or poly(ethylene glycol) self-assembled monolayers onto a glass substrate. Densely packed layers of assembled protein complexes were observed with atomic force microscopy. The protein complexes attached selectively to the amino-terminated regions by electrostatic interactions. Spectral images generated with a hybrid scanning probe and fluorescence microscope confirmed that the patterned proteins retained their native optical signatures.

Ferrochelatase from Rhodopseudomonas sphaeroides: substrate specificity and role of sulfhydryl and arginyl residues

International Nuclear Information System (INIS)

Dailey, H.A.; Fleming, J.E.; Harbin, B.M.

1986-01-01

Purified ferrochelatase from the bacterium Rhodopseudomonas sphaeroides was examined to determine the roles of cationic and sulfhydryl residues in substrate binding. Reaction of the enzyme sulfhydryl residues with N-ethylmaleimide or monobromobimane resulted in a rapid loss of enzyme activity. Ferrous iron, but not porphyrin substrate, had a protective effect against inactivation by these two reagents. Quantitation with 3 H-labeled N-ethylmaleimide revealed that inactivation required one to two sulfhydryl groups to be modified. Modification of arginyl residues with either 2,3-butanedione or camphorquinone 10-sulfonate resulted in a loss of ferrochelatase activity. A kinetic analysis of the modified enzyme showed that the K/sub m/ for ferrous iron was not altered but that the K/sub m/ for the prophyrin substrate was increased. These data suggested that arginyl residues may be involved in porphyrin binding, possibly via charge pair interactions between the arginyl residue and the anionic porphyrin propionate side chain. Modification of lysyl residues had no effect on enzyme activity. The authors also examined the ability of bacterial ferrochelatase to use various 2,4-disubstituted porphyrins as substrates. The authors found that 2,4-bis-acetal- and 2,4-disulfonate deuteroporphyrins were effective substrates for the purified bacterial enzyme and that N-methylprotoporphyrin was an effective inhibitor of the enzyme. Data for the ferrochelatase of R. sphaeroides are compared with previously published data for the eucaryotic enzyme

Hypocholesterolemic Response to Karaya Saponin and Rhodobacter capsulatus in Broiler Chickens

OpenAIRE

Afrose, Sadia; Hossain, Md. Sharoare; Maki, Takaaki; Tsujii, Hirotada

2010-01-01

Dietary karaya saponin and Rhodobacter capsulatus (R. capsulatus) are known to have hypocholesterolemic actions, as reported in our previous studies. This study examined possible synergistic hypocholesterolemic effects of karaya saponin and R. capsulatus in broilers. A total of 150 broilers were allocated into 10 treatments: control, saponin 25 mg, saponin 50 mg, saponin 75 mg, saponin 25 mg+R. capsulatus 0.2 g, saponin 25 mg+R. capsulatus 0.4 g, saponin 50 mg+R. capsulatus 0.2 g, saponin 50 ...

Atomic force microscopy reveals multiple patterns of antenna organization in purple bacteria: implications for energy transduction mechanisms and membrane modeling.

Science.gov (United States)

Sturgis, James N; Niederman, Robert A

2008-01-01

Recent topographs of the intracytoplasmic membrane (ICM) of purple bacteria obtained by atomic force microscopy (AFM) have provided the first surface views of the native architecture of a multicomponent biological membrane at submolecular resolution, representing an important landmark in structural biology. A variety of species-dependent, closely packed arrangements of light-harvesting (LH) complexes was revealed: the most highly organized was found in Rhodobacter sphaeroides in which the peripheral LH2 antenna was seen either in large clusters or in fixed rows interspersed among ordered arrays of dimeric LH1-reaction center (RC) core complexes. A more random organization was observed in other species containing both the LH1 and LH2 complexes, as typified by Rhododspirillum photometricum with randomly packed monomeric LH1-RC core complexes intermingled with large, paracrystalline domains of LH2 antenna. Surprisingly, no structures that could be identified as the ATP synthase or cytochrome bc (1) complexes were observed, which may reflect their localization at ICM vesicle poles or in curved membrane areas, out of view from the flat regions imaged by AFM. This possible arrangement of energy transducing complexes has required a reassessment of energy tranduction mechanisms which place the cytochrome bc (1) complex in close association with the RC. Instead, more plausible proposals must account for the movement of quinone redox species over considerable membrane distances on appropriate time scales. AFM, together with atomic resolution structures are also providing the basis for molecular modeling of the ICM that is leading to an improved picture of the supramolecular organization of photosynthetic complexes, as well as the forces that drive their segregation into distinct domains.

Cytochrome oxidase assembly does not require catalytically active cytochrome C.

Science.gov (United States)

Barrientos, Antoni; Pierre, Danielle; Lee, Johnson; Tzagoloff, Alexander

2003-03-14

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.

Phosphoenolpyruvate-Dependent Fructose Phosphotransferase System of Rhodopseudomonas sphaeroides : Purification and Physicochemical and Immunochemical Characterization of a Membrane-Associated Enzyme I

NARCIS (Netherlands)

Brouwer, Marius; Elferink, Marieke G.L.; Robillard, George T.

1982-01-01

The phosphotransferase system (PTS) of the phototrophic bacterium Rhodopseudomonas sphaeroides consists of a component located in the cytoplasmic membrane and a membrane-associated enzyme called “soluble factor” (SF). SF has been partially purified by a combination of hydrophobic interaction and

Communication: Coherences observed in vivo in photosynthetic bacteria using two-dimensional electronic spectroscopy

International Nuclear Information System (INIS)

Dahlberg, Peter D.; Norris, Graham J.; Wang, Cheng; Viswanathan, Subha; Singh, Ved P.; Engel, Gregory S.

2015-01-01

Energy transfer through large disordered antenna networks in photosynthetic organisms can occur with a quantum efficiency of nearly 100%. This energy transfer is facilitated by the electronic structure of the photosynthetic antennae as well as interactions between electronic states and the surrounding environment. Coherences in time-domain spectroscopy provide a fine probe of how a system interacts with its surroundings. In two-dimensional electronic spectroscopy, coherences can appear on both the ground and excited state surfaces revealing detailed information regarding electronic structure, system-bath coupling, energy transfer, and energetic coupling in complex chemical systems. Numerous studies have revealed coherences in isolated photosynthetic pigment-protein complexes, but these coherences have not been observed in vivo due to the small amplitude of these signals and the intense scatter from whole cells. Here, we present data acquired using ultrafast video-acquisition gradient-assisted photon echo spectroscopy to observe quantum beating signals from coherences in vivo. Experiments were conducted on isolated light harvesting complex II (LH2) from Rhodobacter sphaeroides, whole cells of R. sphaeroides, and whole cells of R. sphaeroides grown in 30% deuterated media. A vibronic coherence was observed following laser excitation at ambient temperature between the B850 and the B850 ∗ states of LH2 in each of the 3 samples with a lifetime of ∼40-60 fs

Communication: Coherences observed in vivo in photosynthetic bacteria using two-dimensional electronic spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Dahlberg, Peter D. [Graduate Program in the Biophysical Sciences, Institute for Biophysical Dynamics, and The James Franck Institute, The University of Chicago, Chicago, Illinois 60637 (United States); Norris, Graham J.; Wang, Cheng; Viswanathan, Subha; Singh, Ved P.; Engel, Gregory S., E-mail: gsengel@uchicago.edu [Department of Chemistry, Institute for Biophysical Dynamics, and The James Franck Institute, The University of Chicago, Chicago, Illinois 60637 (United States)

2015-09-14

Energy transfer through large disordered antenna networks in photosynthetic organisms can occur with a quantum efficiency of nearly 100%. This energy transfer is facilitated by the electronic structure of the photosynthetic antennae as well as interactions between electronic states and the surrounding environment. Coherences in time-domain spectroscopy provide a fine probe of how a system interacts with its surroundings. In two-dimensional electronic spectroscopy, coherences can appear on both the ground and excited state surfaces revealing detailed information regarding electronic structure, system-bath coupling, energy transfer, and energetic coupling in complex chemical systems. Numerous studies have revealed coherences in isolated photosynthetic pigment-protein complexes, but these coherences have not been observed in vivo due to the small amplitude of these signals and the intense scatter from whole cells. Here, we present data acquired using ultrafast video-acquisition gradient-assisted photon echo spectroscopy to observe quantum beating signals from coherences in vivo. Experiments were conducted on isolated light harvesting complex II (LH2) from Rhodobacter sphaeroides, whole cells of R. sphaeroides, and whole cells of R. sphaeroides grown in 30% deuterated media. A vibronic coherence was observed following laser excitation at ambient temperature between the B850 and the B850{sup ∗} states of LH2 in each of the 3 samples with a lifetime of ∼40-60 fs.

Self-trapped excitons in LH2 bacteriochlorophyll-protein complexes under high pressure

International Nuclear Information System (INIS)

Timpmann, K.; Ellervee, Aleksandr; Kuznetsov, Anatoli; Laisaar, Arlentin; Trinkunas, Gediminas; Freiberg, Arvi

2003-01-01

The absorption and emission spectra of excitons in LH2 antenna complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides have been studied under hydrostatic pressure. The measurements made between ambient pressure and 6 kbar over a broad temperature range reveal largely different rates of the pressure-induced shifts for the absorption and emission bands. Numerical calculations based on exciton polaron model provide evidence for the exciton self-trapping at ambient pressure as well as for the pressure stabilization of the self-trapped exciton states responsible for the emission, whereas the light absorbing states belong to nearly free excitons over the whole pressure and temperature ranges studied

A mixed incoherent feed-forward loop contributes to the regulation of bacterial photosynthesis genes.

Science.gov (United States)

Mank, Nils N; Berghoff, Bork A; Klug, Gabriele

2013-03-01

Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes. In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand. Recently, we identified a mixed incoherent feed-forward loop comprising the transcription factor PrrA, the sRNA PcrZ and photosynthesis target genes as part of this regulatory network. This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail.

The mechanisms of protection of antioxidants on Nostoc sphaeroides against UV-B radiation

Science.gov (United States)

Wang, G. H.

UV radiation is one of space harmful factor for earth organisms in space exploration In the present work we studied on the role of antioxidant system in Nostoc sphaeroides K u tz Cyanobacteria and the effects of exogenous antioxidant molecules on its photosynthetic rate under UV-B radiation It was found that UV-B radiation decreased the photosynthetic activity of cyanobacterium but promoted the activity of antioxidant system to protect photosystem II PSII and exogenous antioxidant sodium nitroprusside SNP N-acetylcysteine NAC had an obvious protection on PSII activity under UV-B radiation The activity of SOD Superoxide Dismutase EC 1 15 1 1 CAT Catalase EC 1 11 1 6 POD Peroxidase EC 1 11 1 7 and content of MDA and ASC were improved by 0 5mM and 1mM SNP but 0 1mM SNP decreased the activity of antioxide system Exogenous NAC addition decreased the activity of SOD POD CAT and the content MDA and ASC but exogenous NAC addition increased the content of GSH The results suggested that exogenous SNP and NAC may protect algae by different mechanisms in which SNP maybe play double roles as sources of reactive free radicals or ROS scavengers in formation of algae s protection of PSII under UV-B radiation while NAC does function as antioxidant reagent or precursor of glutathione which could protect PSII directly from UV-B radiation Keyword antioxidant system exogenous or endogenous antioxidant Nostoc sphaeroides photosynthesis UV-B radiation

Visualizing changes in electron distribution in coupled chains of cytochrome bc(1) by modifying barrier for electron transfer between the FeS cluster and heme c(1).

Science.gov (United States)

Cieluch, Ewelina; Pietryga, Krzysztof; Sarewicz, Marcin; Osyczka, Artur

2010-02-01

Cytochrome c(1) of Rhodobacter (Rba.) species provides a series of mutants which change barriers for electron transfer through the cofactor chains of cytochrome bc(1) by modifying heme c(1) redox midpoint potential. Analysis of post-flash electron distribution in such systems can provide useful information about the contribution of individual reactions to the overall electron flow. In Rba. capsulatus, the non-functional low-potential forms of cytochrome c(1) which are devoid of the disulfide bond naturally present in this protein revert spontaneously by introducing a second-site suppression (mutation A181T) that brings the potential of heme c(1) back to the functionally high levels, yet maintains it some 100 mV lower from the native value. Here we report that the disulfide and the mutation A181T can coexist in one protein but the mutation exerts a dominant effect on the redox properties of heme c(1) and the potential remains at the same lower value as in the disulfide-free form. This establishes effective means to modify a barrier for electron transfer between the FeS cluster and heme c(1) without breaking disulfide. A comparison of the flash-induced electron transfers in native and mutated cytochrome bc(1) revealed significant differences in the post-flash equilibrium distribution of electrons only when the connection of the chains with the quinone pool was interrupted at the level of either of the catalytic sites by the use of specific inhibitors, antimycin or myxothiazol. In the non-inhibited system no such differences were observed. We explain the results using a kinetic model in which a shift in the equilibrium of one reaction influences the equilibrium of all remaining reactions in the cofactor chains. It follows a rather simple description in which the direction of electron flow through the coupled chains of cytochrome bc(1) exclusively depends on the rates of all reversible partial reactions, including the Q/QH2 exchange rate to/from the catalytic sites

Two dimensional crystals of LH2 light-harvesting complexes from Ectothiorhodospira sp. and Rhodobacter capsulatus investigated by electron microscopy

NARCIS (Netherlands)

Oling, Frank; Boekema, EJ; deZarate, IO; Visschers, R; vanGrondelle, R; Keegstra, W; Brisson, A; Picorel, R

1996-01-01

Two-dimensional crystals of LH2 (B800-850) light-harvesting complexes from Ectothiorhodospira sp, and Rhodobacter capsulatus were obtained by reconstitution of purified protein into phospholipid vesicles and characterized by electron microscopy. The size of the crystals was up to several

Two-dimensional crystals of LH2 light-harvesting complexes from Ectothiorhodospira sp. and Rhodobacter capsulatus investigated by electron microscopy.

NARCIS (Netherlands)

Oling, F.; Boekema, E.J.; Ortiz de Zarate, I.; Visschers, R.W.; van Grondelle, R.; Keegstra, W.; Brisson, A.; Picorel, R.

1996-01-01

Two-dimensional crystals of LH2 (B800-850) light-harvesting complexes from Ectothiorhodospira sp. and Rhodobacter capsulatus were obtained by reconstitution of purified protein into phospholipid vesicles and characterized by electron microscopy. The size of the crystals was up to several

Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

Science.gov (United States)

Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

2014-10-01

In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome. Copyright © 2014 Elsevier B.V. All rights reserved.

Mechanisms for hydrogen production by different bacteria during mixed-acid and photo-fermentation and perspectives of hydrogen production biotechnology.

Science.gov (United States)

Trchounian, Armen

2015-03-01

H2 has a great potential as an ecologically-clean, renewable and capable fuel. It can be mainly produced via hydrogenases (Hyd) by different bacteria, especially Escherichia coli and Rhodobacter sphaeroides. The operation direction and activity of multiple Hyd enzymes in E. coli during mixed-acid fermentation might determine H2 production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating the activity of different Hyd enzymes is an effective way to enhance H2 production by E. coli in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H2 production. Mixed carbon (sugar and glycerol) utilization studies enlarge the kind of organic wastes used in biotechnology. During photo-fermentation under limited nitrogen conditions, H2 production by Rh. sphaeroides is observed when carbon and nitrogen sources are supplemented. The relationship of H2 production with H(+) transport across the membrane and membrane-associated ATPase activity is shown. On the other hand, combination of carbon sources (succinate, malate) with different nitrogen sources (yeast extract, glutamate, glycine) as well as different metal (Fe, Ni, Mg) ions might regulate H2 production. All these can enhance H2 production yield by Rh. sphaeroides in biotechnology Finally, two of these bacteria might be combined to develop and consequently to optimize two stages of H2 production biotechnology with high efficiency transformation of different organic sources.

Identification of protein W, the elusive sixth subunit of the Rhodopseudomonas palustris reaction center-light harvesting 1 core complex.

Science.gov (United States)

Jackson, Philip J; Hitchcock, Andrew; Swainsbury, David J K; Qian, Pu; Martin, Elizabeth C; Farmer, David A; Dickman, Mark J; Canniffe, Daniel P; Hunter, C Neil

2018-02-01

The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc 1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

A disordered polaron model for polarized fluorescence excitation spectra of LH1 and LH2 bacteriochlorophyll antenna aggregates

International Nuclear Information System (INIS)

Trinkunas, Gediminas; Freiberg, Arvi

2006-01-01

Excitonic polarons in antenna complexes are subject to static lattice disorder. A model has been developed to analyze polarized fluorescence excitation spectra of circular light-harvesting complexes from purple photosynthetic bacteria containing bacteriochlorophyll as the main photoactive pigment that includes both diagonal (energetic) and off-diagonal (structural) disorders. Essential differences of disorder realizations seem to exist between the core LH1 and peripheral LH2 complexes from the bacterium Rhodobacter sphaeroides. The disorder in LH1 appears to be dominated by the structural disorder, while that in LH2, by energetic one. These differences may be due to relatively bigger size of the LH1 complex and, consequently, with its enhanced structural flexibility

Tuning cofactor redox potentials: the 2-methoxy dihedral angle generates a redox potential difference of >160 mV between the primary (Q(A)) and secondary (Q(B)) quinones of the bacterial photosynthetic reaction center.

Science.gov (United States)

Taguchi, Alexander T; Mattis, Aidas J; O'Malley, Patrick J; Dikanov, Sergei A; Wraight, Colin A

2013-10-15

Only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from Rhodobacter sphaeroides. (13)C hyperfine sublevel correlation measurements of the 2-methoxy in the semiquinone states, SQA and SQB, were compared with quantum mechanics calculations of the (13)C couplings as a function of the dihedral angle. X-ray structures support dihedral angle assignments corresponding to a redox potential gap (ΔEm) between QA and QB of ~180 mV. This is consistent with the failure of a ubiquinone analogue lacking the 2-methoxy to function as QB in mutant reaction centers with a ΔEm of ≈160-195 mV.

Exploration of the hydrogen producing potential of Rhodobacter capsulatus chemostat cultures: The application of deceleration-stat and gradient-stat methodology

NARCIS (Netherlands)

Hoekema, S.; Breukelen, van F.R.; Janssen, M.G.J.; Tramper, J.; Wijffels, R.H.

2009-01-01

In this work, the dependency of the volumetric hydrogen production rate of ammonium-limited Rhodobacter capsulatus chemostat cultures on their imposed biomass concentration and dilution rate was investigated. A deceleration-stat experiment was performed by lowering the dilution rate from 1.0 d-1 to

Early Bacteriopheophytin Reduction in Charge Separation in Reaction Centers of Rhodobacter sphaeroides

NARCIS (Netherlands)

Zhu, J.; van Stokkum, I.H.M.; Paparelli, L.; Jones, M.R.; Groot, M.L.

2013-01-01

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and

Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase

NARCIS (Netherlands)

Muntyan, M.S.; Cherepanov, D.A.; Malinen, A.M.; Bloch, D.A.; Sorokin, D.Y.; Severina, I.I.; Ivashina, T.V.; Lahti, R.; Muyzer, G.; Skulachev, V.P.

2015-01-01

Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which

Evidence for a Very Early Intermediate in Bacterial Photosynthesis. A Photon-Echo and Hole-Burning Study of the Primary Donor Band in Rhodopseudomonas Sphaeroides

NARCIS (Netherlands)

Meech, S.R.; Hoff, A.J.

1985-01-01

Two coherent spectroscopic methods, accumulated photon echo and population bottleneck hole-burning, have been employed in a study of the decay rate of the primary donor (P) of Rhodopseudomonas sphaeroides at 1.5 K. The decay rate is instrument-limited in the photon-echo experiment, implying a

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

Science.gov (United States)

Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

2012-10-02

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

Interface for Light-Driven Electron Transfer by Photosynthetic Complexes Across Block Copolymer Membranes.

Science.gov (United States)

Kuang, Liangju; Olson, Tien L; Lin, Su; Flores, Marco; Jiang, Yunjiang; Zheng, Wan; Williams, JoAnn C; Allen, James P; Liang, Hongjun

2014-03-06

Incorporation of membrane proteins into nanodevices to mediate recognition and transport in a collective and scalable fashion remains a challenging problem. We demonstrate how nanoscale photovoltaics could be designed using robust synthetic nanomembranes with incorporated photosynthetic reaction centers (RCs). Specifically, RCs from Rhodobacter sphaeroides are reconstituted spontaneously into rationally designed polybutadiene membranes to form hierarchically organized proteopolymer membrane arrays via a charge-interaction-directed reconstitution mechanism. Once incorporated, the RCs are fully active for prolonged periods based upon a variety of spectroscopic measurements, underscoring preservation of their 3D pigment configuration critical for light-driven charge transfer. This result provides a strategy to construct solar conversion devices using structurally versatile proteopolymer membranes with integrated RC functions to harvest broad regions of the solar spectrum.

Two-photon excitation spectroscopy of carotenoid-containing and carotenoid-depleted LH2 complexes from purple bacteria.

Science.gov (United States)

Stepanenko, Ilya; Kompanetz, Viktor; Makhneva, Zoya; Chekalin, Sergey; Moskalenko, Andrei; Razjivin, Andrei

2009-08-27

We applied two-photon fluorescence excitation spectroscopy to LH2 complex from purple bacteria Allochromatium minutissimum and Rhodobacter sphaeroides . Bacteriochlorophyll fluorescence was measured under two-photon excitation of the samples within the 1200-1500 nm region. Spectra were obtained for both carotenoid-containing and -depleted complexes of each bacterium to allow their direct comparison. The depletion of carotenoids did not alter the two-photon excitation spectra of either bacteria. The spectra featured a wide excitation band around 1350 nm (2x675 nm, 14,800 cm(-1)) which strongly resembled two-photon fluorescence excitation spectra of similar complexes published by other authors. We consider obtained experimental data to be evidence of direct two-photon excitation of bacteriochlorophyll excitonic states in this spectral region.

Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

Directory of Open Access Journals (Sweden)

Alejandro K. Samhan-Arias

2018-05-01

Full Text Available In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.

Modeling the bacterial photosynthetic reaction center. VII. Full simulation of the intervalence hole-transfer absorption spectrum of the special-pair radical cation

International Nuclear Information System (INIS)

Reimers, Jeffrey R.; Hush, Noel S.

2003-01-01

ENDOR data suggests that the special-pair radical cation P + from Rhodobacter sphaeroides is 68% localized on P L while simple interpretations of FTIR difference spectra based primarily on intensity information, but to some extent also bandwidths, suggest near-complete charge localization. We provide a complete a priori spectral simulation of the spectrum of P + in the range 0-5000 cm-1, including explicit treatment of the high-resolution vibrational transitions, the low-resolution hole-transfer absorption centered at 2700 cm-1, and the resonance with the SHOMO to HOMO transition at 2200 cm-1 that resolve the issues concerning the nature of P + . The description of the vibrational aspects of the problem were taken from results of previous density-functional calculations, and a qualitatively realistic large number of vibrational modes (50 antisymmetric and 18-20 symmetric) were included. To facilitate the calculations, a new representation of the vibronic-coupling Hamiltonian for intervalence hole-transfer or electron-transfer problems is introduced, allowing the spectrum to be simulated efficiently using only up to 4x10 9 vibronic basis functions and leading also to new general analytical relationships. Observed spectra are fitted using seven adjustable chemical parameters describing the interactions between the four electronic states involved. The resulting fits provide unique descriptions of the parameters that are insensitive to the source of the observed spectrum or the nature of the symmetric modes used in the model, and all fitted parameters are found to be close in value to those from independent estimates. We determine the electronic coupling, antisymmetric-mode reorganization energy, and redox asymmetry to be J=0.126±0.002 eV, λ=0.139±0.003 eV, and E 0 =0.069±0.002 eV, respectively. Our description forms the basis of understanding for a wide range of other properties observed for Rhodobacter sphaeroides mutants, as well as the properties of the

Absorbance changes accompanying the fast fluorescence induction in the purple bacterium Rhodobacter sphaeroides

Czech Academy of Sciences Publication Activity Database

Bína, David; Litvín, Radek; Vácha, František

2010-01-01

Roč. 105, č. 2 (2010), s. 115-121 ISSN 0166-8595 R&D Projects: GA AV ČR IAA608170603 Institutional research plan: CEZ:AV0Z50510513 Keywords : Photosynthesis * Charge separation * Membrane potential Subject RIV: CE - Biochemistry Impact factor: 2.410, year: 2010

Chemical proprieties of the iron-quinone complex in mutated reaction centers of Rb. sphaeroides

International Nuclear Information System (INIS)

Hałas, Agnieszka; Derrien, Valerie; Sebban, Pierre; Matlak, Krzysztof; Korecki, Józef; Kruk, Jerzy; Burda, Kvĕtoslava

2012-01-01

We investigated type II bacterial photosynthetic reaction centers, which contain a quinone - iron complex (Q A -Fe-Q B ) on their acceptor side. Under physiological conditions it was observed mainly in a reduced high spin state but its low spin ferrous states were also observed. Therefore, it was suggested that it might regulate the dynamical properties of the iron–quinone complex and the protonation and deprotonation events in its neighbourhood. In order to get insight into the molecular mechanism of the NHFe low spin state formation, we preformed Mössbauer studies of a wild type of Rb. sphaeroides and its two mutated forms. Our Mössbauer measurements show that the hydrophobicity of the Q A binding site can be crucial for stabilization of the high spin ferrous state of NHFe.

Potential of Rhodobacter capsulatus Grown in Anaerobic-Light or Aerobic-Dark Conditions as Bioremediation Agent for Biological Wastewater Treatments

OpenAIRE

Stefania Costa; Saverio Ganzerli; Irene Rugiero; Simone Pellizzari; Paola Pedrini; Elena Tamburini

2017-01-01

The use of microorganisms to clean up wastewater provides a cheaper alternative to the conventional treatment plant. The efficiency of this method can be improved by the choice of microorganism with the potential of removing contaminants. One such group is photosynthetic bacteria. Rhodobacter capsulatus is a purple non-sulfur bacterium (PNSB) found to be capable of different metabolic activities depending on the environmental conditions. Cell growth in different media and conditions was teste...

Production and characterization of yeast cytochrome c antibodies; immunological studies of mutants with altered cytochrome c synthesis

International Nuclear Information System (INIS)

Matner, R.R.

1980-01-01

Mutations at the structural gene, CYC1, for iso-1-cytochrome c and at the structural gene, CYC7, for iso-2-cytochrome c can reduce the levels of the respective proteins by varying degrees in Saccharomyces cerevisiae. Mutations at two other loci, cyc2 and cyc3, that are unlinked to either of the structural genes, specifically reduced the levels of both iso-cytochromes c. The cyc2 mutations can cause as low as 10 to 20% of the normal level and cyc3 mutations can cause complete deficiencies. We have explored the possiblity that the CYC2 and CYC3 loci code for maturation functions in the biosynthesis of cytochrome c. The approach used to characterize the nature of the cyc2 and cyc3 induced deficiencies of cytochrome c involved four steps. The results were used to propose possible roles for the CYC2 and CYC3 encoded functions. The CYC3 encoded function is hypothesized to be enzymatic heme attachment. CYC2 may code for a protein that binds and transports apo-cytochrome c through the outer mitochondrial membrane and/or enhances the activity of the heme attachment enzyme

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

Science.gov (United States)

Papa, S; Lorusso, M; Izzo, G; Capuano, F

1981-02-15

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi

Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

OpenAIRE

Hahn, F M; Baker, J A; Poulter, C D

1996-01-01

Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic...

Some experiments on the primary electron acceptor in reaction centres from Rhodopseudomanas sphaeroides

Energy Technology Data Exchange (ETDEWEB)

Wraight, C A; Cogdell, R J; Clayton, R K

1975-01-01

The bacterial reaction center absorbance change at 450 nm (A-450), assigned to an anionic semiquinone, has been suggested as a candidate for the reduced form of the primary electron acceptor in bacterial photosynthesis. In reaction centers of Rhodopseudomonas sphaeroides we have found kinetic discrepancies between the decay of A-450 and the recovery of photochemical competence. In addition, no proton uptake is measurable on the first turnover, although subsequent ones elicit one proton bound per electron. These results are taken to indicate that the acceptor reaction after a long dark period may be different for the first turnover than for subsequent ones. It is suggested that A-450 is still a likely candidate for the acceptor function but that in reaction centers, additional quinone may act as an adventitious primary acceptor when the ''true'' primary acceptor is reduced. Alternatively, the primary acceptor may act in a ''ping-pong'' fashion with respect to subsequent photoelectrons.

Calcium transport in vesicles energized by cytochrome oxidase

Energy Technology Data Exchange (ETDEWEB)

Rosier, Randy N. [Univ. of Rochester, NY (United States)

1979-01-01

Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K⁺ selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K⁺ flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interaction with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.

Directed formation of micro- and nanoscale patterns of functional light-harvesting LH2 complexes.

Science.gov (United States)

Reynolds, Nicholas P; Janusz, Stefan; Escalante-Marun, Maryana; Timney, John; Ducker, Robert E; Olsen, John D; Otto, Cees; Subramaniam, Vinod; Leggett, Graham J; Hunter, C Neil

2007-11-28

The precision placement of the desired protein components on a suitable substrate is an essential prelude to any hybrid "biochip" device, but a second and equally important condition must also be met: the retention of full biological activity. Here we demonstrate the selective binding of an optically active membrane protein, the light-harvesting LH2 complex from Rhodobacter sphaeroides, to patterned self-assembled monolayers at the micron scale and the fabrication of nanometer-scale patterns of these molecules using near-field photolithographic methods. In contrast to plasma proteins, which are reversibly adsorbed on many surfaces, the LH2 complex is readily patterned simply by spatial control of surface polarity. Near-field photolithography has yielded rows of light-harvesting complexes only 98 nm wide. Retention of the native optical properties of patterned LH2 molecules was demonstrated using in situ fluorescence emission spectroscopy.

Cellulose synthases: new insights from crystallography and modeling.

Science.gov (United States)

Slabaugh, Erin; Davis, Jonathan K; Haigler, Candace H; Yingling, Yaroslava G; Zimmer, Jochen

2014-02-01

Detailed information about the structure and biochemical mechanisms of cellulose synthase (CelS) proteins remained elusive until a complex containing the catalytic subunit (BcsA) of CelS from Rhodobacter sphaeroides was crystalized. Additionally, a 3D structure of most of the cytosolic domain of a plant CelS (GhCESA1 from cotton, Gossypium hirsutum) was produced by computational modeling. This predicted structure contributes to our understanding of how plant CelS proteins may be similar and different as compared with BcsA. In this review, we highlight how these structures impact our understanding of the synthesis of cellulose and other extracellular polysaccharides. We show how the structures can be used to generate hypotheses for experiments testing mechanisms of glucan synthesis and translocation in plant CelS. Copyright © 2013 Elsevier Ltd. All rights reserved.

NMR comparison of prokaryotic and eukaryotic cytochromes c

International Nuclear Information System (INIS)

Chau, Meihing; Cai, Meng Li; Timkovich, R.

1990-01-01

1 H NMR spectroscopy has been used to examine ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429) over the pH range 3.5-10.6 and the temperature range 4-60 degree C. Resonance assignments are proposed for main-chain and side-chain protons. Comparison of results for cytochrome c-551 to recently assigned spectra for horse cytochrome c and mutants of yeast iso-1 cytochrome reveals some unique resonances with unusual chemical shifts in all cytochromes that may serve as markers for the heme region. Results for cytochrome c-551 indicate that in the smaller prokaryotic cytochrome, all benzoid side chains are rapidly flipping on the NMR time scale. In contrast, in eukaryotic cytochromes there are some rings flipping slowly on the NMR time scale. The ferrocytochrome c-551 undergoes a transition linked to pH with a pK around 7. The pH behavior of assigned resonances provides evidence that the site of protonation is the inner or buried 17-propionic acid heme substituent (IUPAC-IUB porphyrin nomenclature). Conformational heterogeneity has been observed for segments near the inner heme propionate substituent

Crystallization of a flavodoxin involved in nitrogen fixation in Rhodobacter capsulatus

Energy Technology Data Exchange (ETDEWEB)

Pérez-Dorado, Inmaculada [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Bortolotti, Ana; Cortez, Néstor [Instituto de Biología Molecular y Celular de Rosario (Universidad Nacional de Rosario y CONICET), Suipacha 531, S2002LRK Rosario (Argentina); Hermoso, Juan A., E-mail: xjuan@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain)

2008-05-01

The flavodoxin NifF from R. capsulatus, a candidate for nitrogenase reduction during nitrogen fixation, has been crystallized using the hanging-drop vapour-diffusion method. Preliminary X-ray data processing at 2.17 Å resolution allowed determination of the crystal system and unit-cell parameters. Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 66.49, c = 121.32 Å. X-ray data sets have been collected to 2.17 Å resolution.

Crystallization of a flavodoxin involved in nitrogen fixation in Rhodobacter capsulatus

International Nuclear Information System (INIS)

Pérez-Dorado, Inmaculada; Bortolotti, Ana; Cortez, Néstor; Hermoso, Juan A.

2008-01-01

The flavodoxin NifF from R. capsulatus, a candidate for nitrogenase reduction during nitrogen fixation, has been crystallized using the hanging-drop vapour-diffusion method. Preliminary X-ray data processing at 2.17 Å resolution allowed determination of the crystal system and unit-cell parameters. Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P4 1 2 1 2, with unit-cell parameters a = b = 66.49, c = 121.32 Å. X-ray data sets have been collected to 2.17 Å resolution

ADP-ribosylation of dinitrogenase reductase in Rhodobacter capsulatus

International Nuclear Information System (INIS)

Jouanneau, Y.; Roby, C.; Meyer, C.M.; Vignais, P.M.

1989-01-01

In the photosynthetic bacterium Rhodobacter capsulatus, nitrogenase is regulated by a reversible covalent modification of Fe protein or dinitrogenase reductase (Rc2). The linkage of the modifying group to inactive Rc2 was found to be sensitive to alkali and to neutral hydroxylamine. Complete release of the modifying group was achieved by incubation of inactive Rc2 in 0.4 or 1 M hydroxylamine. After hydroxylamine treatment of the Rc2 preparation, the modifying group could be isolated and purified by affinity chromatography and ion-exchange HPLC. The modifying group comigrated with ADP-ribose on both ion-exchange HPLC and thin-layer chromatography. Analyses by 31 P NMR spectroscopy and mass spectrometry provided further evidence that the modifying group was ADP-ribose. The NMR spectrum of inactive Rc2 exhibited signals characteristic of ADP-ribose; integration of these signals allowed calculation of a molar ration ADP-ribose/Rc2 of 0.63. A hexapeptide carrying the ADP-ribose moiety was purified from a subtilisin digest of inactive Rc2. The structure of this peptide, determined by amino acid analysis and sequencing, is Gly-Arg(ADP-ribose)-Gly-Val-Ile-Thr. This structure allows identification of the binding site for ADP-ribose as Arg 101 of the polypeptide chain of Rc2. It is concluded that nitrogenase activity in R. capsulatus is regulated by reversible ADP-ribosylation of a specific arginyl residue of dinitrogenase reductase

Atomic force microscopy studies of native photosynthetic membranes.

Science.gov (United States)

Sturgis, James N; Tucker, Jaimey D; Olsen, John D; Hunter, C Neil; Niederman, Robert A

2009-05-05

In addition to providing the earliest surface images of a native photosynthetic membrane at submolecular resolution, examination of the intracytoplasmic membrane (ICM) of purple bacteria by atomic force microscopy (AFM) has revealed a wide diversity of species-dependent arrangements of closely packed light-harvesting (LH) antennae, capable of fulfilling the basic requirements for efficient collection, transmission, and trapping of radiant energy. A highly organized architecture was observed with fused preparations of the pseudocrystalline ICM of Blastochloris viridis, consiting of hexagonally packed monomeric reaction center light-harvesting 1 (RC-LH1) core complexes. Among strains which also form a peripheral LH2 antenna, images of ICM patches from Rhodobacter sphaeroides exhibited well-ordered, interconnected networks of dimeric RC-LH1 core complexes intercalated by rows of LH2, coexisting with LH2-only domains. Other peripheral antenna-containing species, notably Rhodospirillum photometricum and Rhodopseudomonas palustris, showed a less regular organization, with mixed regions of LH2 and RC-LH1 cores, intermingled with large, paracrystalline domains. The ATP synthase and cytochrome bc(1) complex were not observed in any of these topographs and are thought to be localized in the adjacent cytoplasmic membrane or in inaccessible ICM regions separated from the flat regions imaged by AFM. The AFM images have served as a basis for atomic-resolution modeling of the ICM vesicle surface, as well as forces driving segregation of photosynthetic complexes into distinct domains. Docking of atomic-resolution molecular structures into AFM topographs of Rsp. photometricum membranes generated precise in situ structural models of the core complex surrounded by LH2 rings and a region of tightly packed LH2 complexes. A similar approach has generated a model of the highly curved LH2-only membranes of Rba. sphaeroides which predicts that sufficient space exists between LH2 complexes

Biogenesis of the yeast cytochrome bc1 complex.

Science.gov (United States)

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-01-01

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

Hydrogen gas production by fermentation from various organic wastewater using Clostridium butyricum NCIB 9576 and Rhodopseudomonas sphaeroides E15-1

Energy Technology Data Exchange (ETDEWEB)

Yoon, Young Sue; Kim, Hyun Kyung; Rye, Hye Yeon; Lee, In Gu; Kim, Mi Sun [Biomass Research Team, Korea Institute of Energy Research, Taejeon (Korea)

2000-03-01

Anaerobic fermentation using Clostidium butyricum NCIB 9576, and phto-fermentation using Rhodopseudomonas sphaeroides E15-1 were studied for the production of hydrogen from Makkoli, fruits (orange and apple, watermelon and melon) and Tofu wastewaters. From the Makkoli wastewater, which contained 0.94 g/liter sugars and 2.74 g/liter solubel starch, approximately 49 mM H{sub 2}/liter wastewater was produced during the initial 18h of the anaerobic fermentation with pH control between 6.5-7.0. Several organic acids such as butyric acid, acetic acid, propionic acid, lactic acid and ethanol were also produced. From watermelon and melon wastewater, which contained 43 g/liter sugars, generated about approximately 71 mM H{sub 2}/liter wastewater was produced during the initial 24h of the anaerobic fermentation. Tofu wastewater, pH 6.5, containing 12.6 g/liter soluble starch and 0.74 g/liter sugars, generated about 30mM H{sub 2}/liter wastewater, along with some organic acids, during the initial 24 h of anaerobic fermentation. Makkoli and Tofu wastewaters as substrates for the photo-fermentation by Rhodopseudomonas sphaeroides E15-1 produced approximately 37.9 and 22.2 {mu}M H{sub 2}/ml wastewaters, respectively for 9 days of incubation under the average of 9,000010,000 lux illumination at the surface of reactor using tungsten halogen lamps. Orange and apple wastewater, which contained 93.4 g/l produced approximately 13.1 {mu}M H{sub 2}/ml wastewater only for 2 days of photo-fermentation and the growth of Rhodopseudomonas spnaeroides E15-1 and hydrogen production were stopped. 22 refs, 4 figs., 2 tabs.

Bacterial Survival under Extreme UV Radiation: A Comparative Proteomics Study of Rhodobacter sp., Isolated from High Altitude Wetlands in Chile

Directory of Open Access Journals (Sweden)

Vilma Pérez

2017-06-01

Full Text Available Salar de Huasco, defined as a polyextreme environment, is a high altitude saline wetland in the Chilean Altiplano (3800 m.a.s.l., permanently exposed to the highest solar radiation doses registered in the world. We present here the first comparative proteomics study of a photoheterotrophic bacterium, Rhodobacter sp., isolated from this remote and hostile habitat. We developed an innovative experimental approach using different sources of radiation (in situ sunlight and UVB lamps, cut-off filters (Mylar, Lee filters and a high-throughput, label-free quantitative proteomics method to comprehensively analyze the effect of seven spectral bands on protein regulation. A hierarchical cluster analysis of 40 common proteins revealed that all conditions containing the most damaging UVB radiation induced similar pattern of protein regulation compared with UVA and visible light spectral bands. Moreover, it appeared that the cellular adaptation of Rhodobacter sp. to osmotic stress encountered in the hypersaline environment from which it was originally isolated, might further a higher resistance to damaging UV radiation. Indeed, proteins involved in the synthesis and transport of key osmoprotectants, such as glycine betaine and inositol, were found in very high abundance under UV radiation compared to the dark control, suggesting the function of osmolytes as efficient reactive oxygen scavengers. Our study also revealed a RecA-independent response and a tightly regulated network of protein quality control involving proteases and chaperones to selectively degrade misfolded and/or damaged proteins.

The cytochrome p450 homepage.

Science.gov (United States)

Nelson, David R

2009-10-01

The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

Laue image analysis. Pt. 2

International Nuclear Information System (INIS)

Greenhough, T.J.; Shrive, A.K.

1994-01-01

Many Laue diffraction patterns from crystals of particular biological or chemical interest are of insufficient quality for their analysis to be feasible. In many cases, this is because of pronounced streaking of the spots owing to either large mosaic spread or disorder introduced during reactions in the crystal. Methods for the analysis of exposures exhibiting radial or near-radial streaking are described, along with their application in Laue diffraction studies of form-II crystals of Met-tRNA synthetase and a photosynthetic reaction centre from Rhodobacter sphaeroides. In both cases, variable elliptical radial masking has led to significant improvements in data quality and quantity and exposures that previously were too streaked to process may now be analysed. These masks can also provide circular profiles as a special case for processing high-quality Laue exposures and spatial-overlap deconvolution may be performed using the elliptical or circular masks. (orig.)

Crystallographic snapshot of cellulose synthesis and membrane translocation.

Science.gov (United States)

Morgan, Jacob L W; Strumillo, Joanna; Zimmer, Jochen

2013-01-10

Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

Structure and expression of cytochrome f in an Oenothera plastome mutant.

Science.gov (United States)

Johnson, E M; Sears, B B

1990-06-01

The chloroplast mutant pm7 is one of a number of mutants derived from the plastome mutator (pm) line of Oenothera hookeri, strain Johansen. Immunoblotting showed that this mutant accumulates a protein that is cross-antigenic with cytochrome f, but five kilodaltons larger than the mature wild-type protein. Since cytochrome f is known to be translated on plastid ribosomes as a precursor with an amino-terminal extension, it is proposed that the unprocessed cytochrome f precursor accumulates in pm7. In addition to this precursor-sized cytochrome f protein, some mature-sized cytochrome f was also found in the mutant plastids. The pm7 mutation is inherited in a non-Mendelian fashion; but no alterations in chloroplast DNA restriction patterns, or differences in DNA sequence in the region encoding cytochrome f, were found in a comparison of the wild-type and pm7 chloroplast DNAs. Although the mutant was capable of synthesizing heme, no covalently-bound heme, normally found associated with mature, functional, cytochrome f was detected in the mutant at sizes expected for the presumed precursor, or for mature cytochrome f. These results indicate that the aberrant accumulation of a precursor-sized cytochrome f in pm7 is not due to a lesion directly in the plastid gene encoding cytochrome f, petA, or to a deficiency in the ability of the mutant plastids to synthesize or accumulate heme.

Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1.

Directory of Open Access Journals (Sweden)

Alexandr N Simonov

Full Text Available Cytochrome P450c17 (P450 17A1, CYP17A1 is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.

Investigation on characteristics of 3D printing using Nostoc sphaeroides biomass.

Science.gov (United States)

An, Yan-Jun; Guo, Chao-Fan; Zhang, Min; Zhong, Ze-Ping

2018-06-27

Gel-like constructs can be produced using an extrusion-based 3D food printing (3D-FP) technique. Nostoc sphaeroides biomass is a natural gel material. Considering its good nutrition and rheological properties, these algae were chosen in this study as supply material (ink) for 3D-FP. With this gel material, the extrusion-based 3D printing system was set as a model, and the printing behavior was investigated. Furthermore, the 3D-FP mechanisms were explained through low-field nuclear magnetic resonance and rheological measurements. Results indicated that although fresh biomass gel was printable, nonuniformity and instability occurred during printing. Blanched inks showed nonsmooth printing behavior, which was associated with a decrease in elasticity and viscosity. The printability was improved by increasing the rehydration time to 24 h when rehydrated powder was used. Increasing the rehydration time increased the water-binding degree. Pre-gelatinized potato starch was added to the mixture at ratios ranging from 1 to 100 g kg -1 . The best printing outcome was observed at 40 g kg -1 potato starch. We emphasize that elasticity and viscosity balance is an essential parameter to achieve printability. The strategies adopted in this work provide new insights into the development of personalized food regarding texture and nutritional additive content. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Reduction of reversed micelle entrapped cytochrome c and cytochrome c3 by electrons generated by pulse radiolysis or by pyrene photoionization

International Nuclear Information System (INIS)

Vlsser, A.J.W.G.; Fendler, J.H.

1982-01-01

Horse heart cytochrome c and cytochrome c 3 , isolated from Desulfovibrio vulgaris, have been incorporated in sodium bis(2-ethylhexyl)sulfosuccinate (AOT) entrapped water pools in heptane. The absorption spectra of the cytochromes have been found to be strongly dependent on the water to AOT concentration ratios. The proteins solubilized in heptane by the AOT reversed micelles have retained their ability to mediate electron transfer. They reacted very rapidly with hydrated electrons, generated pulse radiolytically or, alternatively, formed in the laser photoionization of pyrene

Plastocyanin/cytochrome c6 interchange in Scenedesmus vacuolatus.

Science.gov (United States)

Miramar, M Dolores; Inda, Luis A; Saraiva, Lígia M; Peleato, M Luisa

2003-12-01

Plastocyanin and cytochrome c6 from the green alga Scenedesmus vacuolatus were immunoquantified in cells grown under different concentrations of copper and iron. Plastocyanin expression was constitutive, its synthesis was not significantly affected by iron availability, and increases with copper availability. On the contrary, cytochrome c6 synthesis is repressed by copper, and only residual amounts of the protein were detected at 0.1 micromol/L copper. Under copper deficiency, cytochrome c6 is slightly dependent on iron. In natural environments, plastocyanin seems to be the predominant electron donor to P700.

Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states.

Science.gov (United States)

Ogura, T; Yoshikawa, S; Kitagawa, T

1985-12-17

Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).

The SMARTCyp cytochrome P450 metabolism prediction server

DEFF Research Database (Denmark)

Rydberg, Patrik; Gloriam, David Erik Immanuel; Olsen, Lars

2010-01-01

The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism.......The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism....

Magnetic circular dichroism studies on microsomal aryl hydrocarbon hydroxylase: comparison with cytochrome b/sub 5/ and cytochrome P-450/sub cam/

Energy Technology Data Exchange (ETDEWEB)

Vickery, L; Salmon, A; Sauer, K

1975-01-01

Magnetic circular dichroism spectra are reported for the visible and near ultraviolet spectral regions of liver microsomes from dimethylbenzanthracene-treated rats. The sequential addition of NADH, dithionite, and carbon monoxide enables us to determine contributions to the magnetic circular dichroism by cytochromes b/sub 5/ and P-450, which dominate the spectra. The magnetic circular dichroism of the microsomal preparation is compared with that of purified oxidized and reduced cytochrome b/sub 5/ from pig liver and with the camphor-complexed and camphor-free oxidized, reduced, and reduced carbonmonoxy cytochrome P-450/sub cam/ from Pseudomonas putida. The magnetic circular dichroism spectra of the membrane bound cytochrome b/sub 5/ are similar to those of the purified protein, indicating that little or no alteration in the environment of the heme occurs during the isolation procedure. The soluble bacterial cytochrome P-450/sub cam/ also appears to be a suitable model for microsomal P-450, although differences in the magnetic circular dichroism intensity are observed for the two enzymes. No effect of dimethylbenzanthracene on the magnetic circular dichroism spectra of induced compared to control rat microsomes could be observed.

Cytochrome P450 monooxygenases and insecticide resistance in insects.

OpenAIRE

Bergé, J B; Feyereisen, R; Amichot, M

1998-01-01

Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the seque...

Evidence for high-pressure-induced rupture of hydrogen bonds in LH2 photosynthetic antenna pigment-protein complexes

International Nuclear Information System (INIS)

Kangur, L; Leiger, K; Freiberg, A

2008-01-01

The bacteriochlorophyll a-containing LH2 light harvesting complex is an integral membrane protein that catalyzes the photosynthetic process in purple photosynthetic bacteria. The LH2 complexes from Rhodobacter sphaeroides show characteristic strong absorbance at 800 and 850 nm due to the bacteriochlorophyll a molecules confined in two separate areas of the protein. Using these cofactors as intrinsic probes to monitor changes in membrane protein structure, we investigate the response to high hydrostatic pressure up to 2.1 GPa of LH2 complexes embedded into natural membrane environment or extracted with detergent. We demonstrate that high pressure does induce significant alterations to the tertiary structure of the protein in proximity of the protein-bound bacteriochlorophyll a molecules, including breakage of the hydrogen bond they are involved in. The membrane-embedded complexes appear more resilient to damaging effects of the compression than the complexes extracted into detergent environment. This difference has tentatively been explained by more compact structure of the membrane-embedded complexes

Evidence for high-pressure-induced rupture of hydrogen bonds in LH2 photosynthetic antenna pigment-protein complexes

Energy Technology Data Exchange (ETDEWEB)

Kangur, L; Leiger, K; Freiberg, A [Institute of Physics, University of Tartu, Riia 142, Tartu 51014 (Estonia)

2008-07-15

The bacteriochlorophyll a-containing LH2 light harvesting complex is an integral membrane protein that catalyzes the photosynthetic process in purple photosynthetic bacteria. The LH2 complexes from Rhodobacter sphaeroides show characteristic strong absorbance at 800 and 850 nm due to the bacteriochlorophyll a molecules confined in two separate areas of the protein. Using these cofactors as intrinsic probes to monitor changes in membrane protein structure, we investigate the response to high hydrostatic pressure up to 2.1 GPa of LH2 complexes embedded into natural membrane environment or extracted with detergent. We demonstrate that high pressure does induce significant alterations to the tertiary structure of the protein in proximity of the protein-bound bacteriochlorophyll a molecules, including breakage of the hydrogen bond they are involved in. The membrane-embedded complexes appear more resilient to damaging effects of the compression than the complexes extracted into detergent environment. This difference has tentatively been explained by more compact structure of the membrane-embedded complexes.

One-electron reduction of mitomycin c by rat liver : role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

NARCIS (Netherlands)

Vromans, R M; Van de Straat, R; Groeneveld, M.; Vermeulen, N P

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of

In vitro effects of myricetin, morin, apigenin, (+)-taxifolin, (+)-catechin, (−)-epicatechin, naringenin and naringin on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase

International Nuclear Information System (INIS)

Çelik, Haydar; Koşar, Müberra; Arinç, Emel

2013-01-01

Highlights: • We assessed inhibitory effects of 8 dietary flavonoids on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase. • The flavonol myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC 50 value of 0.35 μM. • We investigated kinetics of myricetin-induced inhibition in detail. • We explored the structure–inhibitory activity relationship of compounds. • Modulation of cytochrome b5 reduction indicates a potential for myricetin to lead to some food–drug/xenobiotic interactions. - Abstract: The microsomal NADH-dependent electron transport system consisting of cytochrome b5 reductase and cytochrome b5 participates in a number of physiologically important processes including lipid metabolism as well as is involved in the metabolism of various drug and xenobiotics. In the present study, we assessed the inhibitory effects of eight dietary flavonoids representing five distinct chemical classes on cytochrome b5 reduction by purified cytochrome b5 reductase. From the flavonoids tested, myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC 50 value of 0.35 μM. Myricetin inhibited b5 reductase noncompetitively with a K i of 0.21 μM with respect to cofactor NADH, and exhibited a non-linear relationship indicating non-Michaelis–Menten kinetic binding with respect to cytochrome b5. In contrast to the potent inhibitory activity of myricetin, (+)-taxifolin was found to be a weak inhibitor (IC 50 = 9.8 μM). The remaining flavonoids were inactive within the concentration range tested (1–50 μM). Analysis of structure–activity data suggested that simultaneous presence of three OH groups in ring B is a primary structural determinant for a potent enzyme inhibition. Our results suggest that inhibition of the activity of this system by myricetin or myricetin containing diets may influence the metabolism of therapeutic drugs as well as detoxification of xenobiotics

Effectiveness of cytochrome C and cepharanthin for leukopenia following multidisciplinary treatment

International Nuclear Information System (INIS)

Tabata, Kumiko; Endow, Masaru; Suzuki, Hirotoshi

1986-01-01

Leukopenia is one of important problems for multidisciplinary treatment of malignant tumor. We could not be able to take a continuous cancer therapy because of leukopenia. And then we had a study of effectiveness combination treatment of cytochrome C with cepharanthin for leukopenia of cancer patient. We carried on the study of 3 classifications of treatment as follows, a) cytochrome C only, b) combined cytochrome C with cepharanthin, and c) control group without drugs. Bone marrow potentiality is individual differentiation and then the group was administrated both cytochrome C and cepharanthin following radiotherapy associated with postoperative breast cancer. The above description lead to conclusion that combination treatment of cytochrome C and cepharanthin was available for protective drugs from multidisciplinary treatment induced leukemia. (author)

The reaction of neuroglobin with potential redox protein partners cytochrome b5  and cytochrome c

DEFF Research Database (Denmark)

Fago, Angela; Mathews, A.J.; Moens, L.

2006-01-01

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b5 is relatively slow (k=6×102M-1s-1 at pH 7.0) and thus is unlikely to be of physiological...... significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2×107M-1s-1) with an apparent overall equilibrium constant of 1μM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis...

Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450

DEFF Research Database (Denmark)

Laursen, Tomas; Jensen, Kenneth; Møller, Birger Lindberg

2011-01-01

The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR und...... to serve as an effective electron transferring "nano-machine"....

In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent

International Nuclear Information System (INIS)

Spano, Anthony J.; Chen, Frank S.; Goodman, Benjamin E.; Sabat, Agnes E.; Simon, Martha N.; Wall, Joseph S.; Correia, John J.; McIvor, Wilson; Newcomb, William W.; Brown, Jay C.; Schnur, Joel M.; Lebedev, Nikolai

2007-01-01

The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass ∼ 4.3 MDa, representing 101 ± 11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring

[Regulation of alternative CO{sub 2} fixation pathways in procaryotic and eucaryotic photosynthetic organisms]. Progress report

Energy Technology Data Exchange (ETDEWEB)

1992-12-31

The major goal of this project is to determine how microorganisms regulate the assimilation of CO{sup 2} via pathways alternative to the usual Calvin reductive pentose phosphate scheme. In particular, we are interest in the molecular basis for switches in CO{sub 2} metabolic paths. Several earlier studies had indicated that purple nonsulfur photosynthetic bacteria assimilate significant amounts of CO{sub 2} via alternative non-Calvin routes. We have deleted the gene that encodes. RubisCo (ribulose bisphosphate carboxylase/oxygenase) in both the Rhodobacter sphaeroids and Rhodospirillum rubrum. The R. sphaeroides RubisCO deletion strain (strain 16) could not grow under photoheterotrophic conditions with malate as electron donor and CO{sub 2} as the electron acceptor; however the R. rub RubisCO deletion strain (strain I-19) could. Over the past year we have sought to physiologically characterize strain 16PHC. We found that, 16PHC exhibited rates of whole-cell CO{sub 2} fixation which were significantly higher than strain 16. Strain 16PHC could not grow photolithoautotrophically in a CO{sub 2} atmosphere; however, CO{sub 2} fixation catalyzed by photoheterotrophically grown 16PHC was repressed by the addition of DMSO. Likewise, we found that cells initially grown in the presence of DMSO could induce the CO{sub 2} fixation system when DMSO was removed. Thus, these results suggested that both PHC and I-19 could be used to study alternative CO{sub 2} fixation reactions and their significance in R. sphaexoides and R. rubrum.

[Regulation of alternative CO[sub 2] fixation pathways in procaryotic and eucaryotic photosynthetic organisms

Energy Technology Data Exchange (ETDEWEB)

1992-01-01

The major goal of this project is to determine how microorganisms regulate the assimilation of CO[sup 2] via pathways alternative to the usual Calvin reductive pentose phosphate scheme. In particular, we are interest in the molecular basis for switches in CO[sub 2] metabolic paths. Several earlier studies had indicated that purple nonsulfur photosynthetic bacteria assimilate significant amounts of CO[sub 2] via alternative non-Calvin routes. We have deleted the gene that encodes. RubisCo (ribulose bisphosphate carboxylase/oxygenase) in both the Rhodobacter sphaeroids and Rhodospirillum rubrum. The R. sphaeroides RubisCO deletion strain (strain 16) could not grow under photoheterotrophic conditions with malate as electron donor and CO[sub 2] as the electron acceptor; however the R. rub RubisCO deletion strain (strain I-19) could. Over the past year we have sought to physiologically characterize strain 16PHC. We found that, 16PHC exhibited rates of whole-cell CO[sub 2] fixation which were significantly higher than strain 16. Strain 16PHC could not grow photolithoautotrophically in a CO[sub 2] atmosphere; however, CO[sub 2] fixation catalyzed by photoheterotrophically grown 16PHC was repressed by the addition of DMSO. Likewise, we found that cells initially grown in the presence of DMSO could induce the CO[sub 2] fixation system when DMSO was removed. Thus, these results suggested that both PHC and I-19 could be used to study alternative CO[sub 2] fixation reactions and their significance in R. sphaexoides and R. rubrum.

Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

Science.gov (United States)

Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

1976-03-01

Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

Potential use of thermophilic dark fermentation effluents in photofermentative hydrogen production by Rhodobacter capsulatus

Energy Technology Data Exchange (ETDEWEB)

Ozgura, E.; Afsar, N.; Eroglu, I. [Middle East Technical University, Department of Chemical Engineering, 06531 Ankara (Turkey); De Vrije, T.; Claassen, P.A.M. [Wageningen UR, Agrotechnology and Food Sciences Group, Wageningen UR, P.O. Box 17, 6700 AA Wageningen (Netherlands); Yucel, M.; Gunduz, U. [Middle East Technical University, Department of Biology, 06531 Ankara (Turkey)

2010-12-15

Biological hydrogen production by a sequential operation of dark and photofermentation is a promising route to produce hydrogen. The possibility of using renewable resources, like biomass and agro-industrial wastes, provides a dual effect of sustainability in biohydrogen production and simultaneous waste removal. In this study, photofermentative hydrogen production on effluents of thermophilic dark fermentations on glucose, potato steam peels (PSP) hydrolysate and molasses was investigated in indoor, batch operated bioreactors. An extreme thermophile Caldicellulosiruptor saccharolyticus was used in the dark fermentation step, and Rhodobacter capsulatus (DSM1710) was used in the photofermentation step. Addition of buffer, Fe and Mo to dark fermentor effluents (DFEs) improved the overall efficiency of hydrogen production. The initial acetate concentration in the DFE needed to be adjusted to 30-40 mM by dilution to increase the yield of hydrogen in batch light-supported fermentations. The thermophilic DFEs are suitable for photofermentative hydrogen production, provided that they are supplemented with buffer and nutrients. The overall hydrogen yield of the two-step fermentations was higher than the yield of single step dark fermentations.

New insight into the mechanism of mitochondrial cytochrome c function

DEFF Research Database (Denmark)

Chertkova, Rita V; Brazhe, Nadezda A; Bryantseva, Tatiana V

2017-01-01

We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76...... with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c....

Influence of polyhalogenated aromatic hydrocarbons on the induction, activity, and stabilization of cytochrome P450

International Nuclear Information System (INIS)

Voorman, R.

1987-01-01

In the course of experiments evaluating the metabolism of polybrominated biphenyls by cytochrome P450 isozymes induced by 3,4,5,3',4',5'-hexabromobiphenyl (HBB), it was discovered that the inducer remained closely associated with cytochrome P450d. Subsequent purification of cytochromes from HBB treated rates revealed a 0.5:1 association of HBB to cytochrome P450d but virtually none with cytochrome P450c or cytochrome b5. Immunochemical quantitation of cytochrome P450d in the same microsomes yielded a ratio of P450d:HBB that approached unity. Measurement of cytochrome P450d estradiol 2-hydroxylase indicated non-competitive or mixed type inhibition caused by HBB at a concentration of 10-1000 nM. Inhibition was specific to cytochrome P450d since estradiol 2-hydroxylase catalyzed by cytochrome P450h was unaffected by HBB. The ability of HCB and isosafrole to stabilize cytochrome P450d, and thus indirectly influence regulation of the enzyme, was evaluated by treating rats with a dose of TCDD sufficient to produce maximum induction of cytochromes P450c and P450d via the Ah receptor, yet insufficient to bind to the enzyme. Subsequent treatment of these animals with HCB or isosafrole and a radiolabeled amino acid, revealed a significant increase in cytochrome P450d specific content relative to cytochrome P450c and significant retention of the radiolabel in P450d relative to rats treated only with TCDD

Field evidence for the potential of Rhodobacter capsulatus as Biofertilizer for flooded rice.

Science.gov (United States)

Gamal-Eldin, Hosny; Elbanna, Khaled

2011-02-01

In a previous study, we evaluated the effects of inoculating rice plants with the phototrophic purple nonsulfur bacterium Rhodobacter capsulatus (Rc) on growth and yield of rice in pots and lysimeter experiments and the results obtained have been highly encouraging. In this study, we carried out two field experiments: one in the experimental farm of the Faculty of Agriculture, Fayoum University, and the second in a farmer's field in Kafr El-sheikh, to assess the effects of Rc on growth and yield of rice in comparison and in combination with chemical nitrogen fertilizer (CNF) and farmyard manure. The results indicated that both biological and grain yields in all the Rc inoculated treatments were significantly higher than those in the uninoculated corresponding treatments in both fields. With regard to grain yield, the major factor for determining the effectiveness of any agricultural treatment, inoculation with Rc in combination with 50% of the recommended CNF rate gave a grain yield that was statistically equivalent to that obtained with 100% of the recommended CNF rate. These results provide a clear evidence for the potential of Rc as biofertilizer for flooded rice under field conditions.

Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.

Science.gov (United States)

Van Pelt, F N; Meierink, Y J; Blaauboer, B J; Weterings, P J

1990-12-01

We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

Mitochondrial cytochrome c biogenesis: no longer an enigma.

Science.gov (United States)

Babbitt, Shalon E; Sutherland, Molly C; San Francisco, Brian; Mendez, Deanna L; Kranz, Robert G

2015-08-01

Cytochromes c (cyt c) and c1 are heme proteins that are essential for aerobic respiration. Release of cyt c from mitochondria is an important signal in apoptosis initiation. Biogenesis of c-type cytochromes involves covalent attachment of heme to two cysteines (at a conserved CXXCH sequence) in the apocytochrome. Heme attachment is catalyzed in most mitochondria by holocytochrome c synthase (HCCS), which is also necessary for the import of apocytochrome c (apocyt c). Thus, HCCS affects cellular levels of cyt c, impacting mitochondrial physiology and cell death. Here, we review the mechanisms of HCCS function and the roles of heme and residues in the CXXCH motif. Additionally, we consider concepts emerging within the two prokaryotic cytochrome c biogenesis pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Solution NMR study of the yeast cytochrome c peroxidase: cytochrome c interaction

Energy Technology Data Exchange (ETDEWEB)

Volkov, Alexander N., E-mail: ovolkov@vub.ac.be; Nuland, Nico A. J. van [Vrije Universiteit Brussel, Jean Jeener NMR Centre, Structural Biology Brussels (Belgium)

2013-07-15

Here we present a solution NMR study of the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP), a paradigm for understanding the biological electron transfer. Performed for the first time, the CcP-observed heteronuclear NMR experiments were used to probe the Cc binding in solution. Combining the Cc- and CcP-detected experiments, the binding interface on both proteins was mapped out, confirming that the X-ray structure of the complex is maintained in solution. Using NMR titrations and chemical shift perturbation analysis, we show that the interaction is independent of the CcP spin-state and is only weakly affected by the Cc redox state. Based on these findings, we argue that the complex of the ferrous Cc and the cyanide-bound CcP is a good mimic of the catalytically-active Cc-CcP compound I species. Finally, no chemical shift perturbations due to the Cc binding at the low-affinity CcP site were observed at low ionic strength. We discuss possible reasons for the absence of the effects and outline future research directions.

Assembly and structural organization of pigment-protein complexes in membranes of Rhodopseudomonas sphaeroides

International Nuclear Information System (INIS)

Hunter, C.N.; Pennoyer, J.D.; Niederman, R.A.

1982-01-01

The B875 and B800-850 light-harvesting pigment-protein complexes of Rhodopseudomonas sphaeroides are characterized further by lithium dodecyl sulfate/polyacrylamide gel electrophoresis at 4 degrees C. Bacteriochlorophyll a was shown in reconstruction studies to remain complexed with its respective binding proteins during this procedure. From distributions in these gels, a quantitative description for the arrangement of the complexes is proposed. Assembly of the complexes was examined in delta-aminolevulinate-requiring mutant H-5 after a shift from high- to low-light intensity. After 10 h of delta-[ 3 H]aminolevulinate labeling, the specific radioactivity of bacteriochlorophyll in a fraction containing putative membrane invaginations reached the maximal level, while that of the mature photosynthetic membrane was at only one-third this level. This suggests that membrane invaginations are sites of preferential bacteriochlorophyll synthesis in which completed pigment-proteins exist transiently. Analysis of the 3 H distribution after electrophoretic separation further suggests that photosynthetic membranes grow mainly by addition of B800-850 to preformed membrane consisting largely of B875 and photochemical reaction centers. These results corroborate the above model for the structural organization of the light-harvesting system and indicate that the structurally and functionally discrete B800-850 pool is not completely assembled until all B875 sites for B800-850 interactions are occupied

Physicochemical Characterization and Functional Analysis of the Polysaccharide from the Edible Microalga Nostoc sphaeroides

Directory of Open Access Journals (Sweden)

Haifeng Li

2018-02-01

Full Text Available Nostoc colonies have been used as food and medicine for centuries, and their main supporting matrix is polysaccharides, which help Nostoc cells resist various environmental stresses including oxidative stress. Here we isolated a polysaccharide, nostoglycan, from cultured Nostoc sphaeroides colonies and determined its physicochemical properties, which revealed a characteristic infrared absorption spectrum typical of polysaccharides and an amorphous morphology with rough surfaces. We also show that nostoglycan has strong moisture absorption and retention capacities and a high relative viscosity. Using Caenorhabditis elegans models, we then demonstrate that nostoglycan is capable of improving overall survival rate of the animals under increased oxidative stress caused by paraquat. Nostoglycan also reduces reactive oxygen species level, inhibits protein carbonyl formation and lipid peroxidation, and increases activities of superoxide dismutase and catalase in paraquat-exposed nematodes. As oxidative stress may drive tumor progression, we further demonstrate that nostoglycan can suppress the proliferation of several types of tumor cells and induce apoptosis of human lung adenocarcinoma A549 cells via caspase-3 activation. Together, our results yield important information on the physicochemical characteristics and demonstrate the antioxidant and anti-proliferative functions of nostoglycan, and thus provide an insight into its potential in food and health industries.

Cytochromes c': Structure, Reactivity and Relevance to Haem-Based Gas Sensing.

Science.gov (United States)

Hough, Michael A; Andrew, Colin R

2015-01-01

Cytochromes c' are a group of class IIa cytochromes with pentacoordinate haem centres and are found in photosynthetic, denitrifying and methanotrophic bacteria. Their function remains unclear, although roles in nitric oxide (NO) trafficking during denitrification or in cellular defence against nitrosoative stress have been proposed. Cytochromes c' are typically dimeric with each c-type haem-containing monomer folding as a four-α-helix bundle. Their hydrophobic and crowded distal sites impose severe restrictions on the binding of distal ligands, including diatomic gases. By contrast, NO binds to the proximal haem face in a similar manner to that of the eukaryotic NO sensor, soluble guanylate cyclase and bacterial analogues. In this review, we focus on how structural features of cytochromes c' influence haem spectroscopy and reactivity with NO, CO and O2. We also discuss the relevance of cytochrome c' to understanding the mechanisms of gas binding to haem-based sensor proteins. © 2015 Elsevier Ltd. All rights reserved.

A cytosolic cytochrome b 5-like protein in yeast cell accelerating the electron transfer from NADPH to cytochrome c catalyzed by Old Yellow Enzyme

International Nuclear Information System (INIS)

Nakagawa, Manabu; Yamano, Toshio; Kuroda, Kiyo; Nonaka, Yasuki; Tojo, Hiromasa; Fujii, Shigeru

2005-01-01

A 410-nm absorbing species which enhanced the reduction rate of cytochrome c by Old Yellow Enzyme (OYE) with NADPH was found in Saccharomyces cerevisiae. It was solubilized together with OYE by the treatment of yeast cells with 10% ethyl acetate. The purified species showed visible absorption spectra in both oxidized and reduced forms, which were the same as those of the yeast microsomal cytochrome b 5 . At least 14 amino acid residues of the N-terminal region coincided with those of yeast microsomal b 5 , but the protein had a lower molecular weight determined to be 12,600 by SDS-PAGE and 9775 by mass spectrometry. The cytochrome b 5 -like protein enhanced the reduction rate of cytochrome c by OYE, and a plot of the reduction rates against its concentration showed a sigmoidal curve with an inflexion point at 6 x 10 -8 M of the protein

The novel cytochrome c6 of chloroplasts: a case of evolutionary bricolage?

Science.gov (United States)

Howe, Christopher J; Schlarb-Ridley, Beatrix G; Wastl, Juergen; Purton, Saul; Bendall, Derek S

2006-01-01

Cytochrome c6 has long been known as a redox carrier of the thylakoid lumen of cyanobacteria and some eukaryotic algae that can substitute for plastocyanin in electron transfer. Until recently, it was widely accepted that land plants lack a cytochrome c6. However, a homologue of the protein has now been identified in several plant species together with an additional isoform in the green alga Chlamydomonas reinhardtii. This form of the protein, designated cytochrome c6A, differs from the 'conventional' cytochrome c6 in possessing a conserved insertion of 12 amino acids that includes two absolutely conserved cysteine residues. There are conflicting reports of whether cytochrome c6A can substitute for plastocyanin in photosynthetic electron transfer. The evidence for and against this is reviewed and the likely evolutionary history of cytochrome c6A is discussed. It is suggested that it has been converted from a primary role in electron transfer to one in regulation within the chloroplast, and is an example of evolutionary 'bricolage'.

Importance of c-Type cytochromes for U(VI reduction by Geobacter sulfurreducens

Directory of Open Access Journals (Sweden)

Leang Ching

2007-03-01

Full Text Available Abstract Background In order to study the mechanism of U(VI reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI with acetate serving as the electron donor was investigated. Results The ability of several c-type cytochrome deficient mutants to reduce U(VI was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60% the ability of G. sulfurreducens to reduce U(VI. Involvement in U(VI reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI reduction. A subpopulation of both wild type and U(VI reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III revealed no correlation between the impact of cytochrome deletion on U(VI reduction and reduction of Fe(III hydroxide and chelated Fe(III. Conclusion This study indicates that c-type cytochromes are involved in U(VI reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium

The amino acid sequence of cytochrome c from Cucurbita maxima L. (pumpkin)

Science.gov (United States)

Thompson, E. W.; Richardson, M.; Boulter, D.

1971-01-01

The amino acid sequence of pumpkin cytochrome c was determined on 2μmol of protein. Some evidence was found for the occurrence of two forms of cytochrome c, whose sequences differed in three positions. Pumpkin cytochrome c consists of 111 residues and is homologous with mitochondrial cytochromes c from other plants. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50005 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1971), 121, 7. PMID:5131733

Aspirin Induces Apoptosis through Release of Cytochrome c from Mitochondria

Directory of Open Access Journals (Sweden)

Katja C. Zimmermann

2000-01-01

Full Text Available Nonsteroidal anti-inflammatory drugs (NSAID reduce the risk for cancer, due to their anti proliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and triggers release of cytochrome c into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.

The Role of Cytochromes P450 in Infection

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Elisavet Stavropoulou

2018-01-01

Full Text Available Cytochromes are expressed in many different tissues of the human body. They are found mostly in intestinal and hepatic tissues. Cytochromes P450 (CYPs are enzymes that oxidize substances using iron and are able to metabolize a large variety of xenobiotic substances. CYP enzymes are linked to a wide array of reactions including and O-dealkylation, S-oxidation, epoxidation, and hydroxylation. The activity of the typical P450 cytochrome is influenced by a variety of factors, such as genus, environment, disease state, herbicide, alcohol, and herbal medications. However, diet seems to play a major role. The mechanisms of action of dietary chemicals, macro- and micronutrients on specific CYP isoenzymes have been extensively studied. Dietary modulation has effects upon the metabolism of xenobiotics. Cytochromes harbor intra- or interindividual and intra- or interethnic genetic polymorphisms. Bacteria were shown to express CYP-like genes. The tremendous metabolic activity of the microbiota is associated to its abundant pool of CYP enzymes, which catalyze phase I and II reactions in drug metabolism. Disease states, intestinal disturbances, aging, environmental toxic effects, chemical exposures or nutrition modulate the microbial metabolism of a drug before absorption. A plethora of effects exhibited by most of CYP enzymes can resemble those of proinflammatory cytokines and IFNs. Moreover, they are involved in the initiation and persistence of pathologic pain by directly activating sensory neurons and inflammatory cytokines.

A web-based resource for the Arabidopsis P450, cytochromes b5, NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases (http://www.P450.kvl.dk).

Science.gov (United States)

Paquette, Suzanne M; Jensen, Kenneth; Bak, Søren

2009-12-01

Gene and genome duplication is a key driving force in evolution of plant diversity. This has resulted in a number of large multi-gene families. Two of the largest multi-gene families in plants are the cytochromes P450 (P450s) and family 1 glycosyltransferases (UGTs). These two families are key players in evolution, especially of plant secondary metabolism, and in adaption to abiotic and biotic stress. In the model plant Arabidopsis thaliana there are 246 and 112 cytochromes P450 and UGTs, respectively. The Arabidopsis P450, cytochromes b(5), NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases website (http://www.P450.kvl.dk) is a sequence repository of manually curated sequences, multiple sequence alignments, phylogenetic trees, sequence motif logos, 3D structures, intron-exon maps, and customized BLAST datasets.

Cytochrome c and c1 heme lyases are essential in Plasmodium berghei.

Science.gov (United States)

Posayapisit, Navaporn; Songsungthong, Warangkhana; Koonyosying, Pongpisid; Falade, Mofolusho O; Uthaipibull, Chairat; Yuthavong, Yongyuth; Shaw, Philip J; Kamchonwongpaisan, Sumalee

Malaria parasites possess a de novo heme synthetic pathway. Interestingly, this pathway is dispensable during the blood stages of development in mammalian hosts. The assembly of the two most important hemeproteins, cytochromes c and c1, is mediated by cytochrome heme lyase enzymes. Plasmodium spp. possess two cytochrome heme lyases encoded by separate genes. Given the redundancy of heme synthesis, we sought to determine if heme lyase function also exhibits redundancy. To answer this question, we performed gene knockout experiments. We found that the PBANKA_143950 and PBANKA_0602600 Plasmodium berghei genes encoding cytochrome c (Pbcchl) and cytochrome c1 (Pbcc 1 hl) heme lyases, respectively, can only be disrupted when a complementary gene is present. In contrast, four genes in the de novo heme synthesis pathway can be disrupted without complementation. This work provides evidence that Pbcchl and Pbcc 1 hl are both essential and thus may be antimalarial targets. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains

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Míriam Cristina Sakuragui Matuo

2010-09-01

Full Text Available Saccharomyces cerevisiae has been widely used in mutagenicity tests due to the presence of a cytochrome P-450 system, capable of metabolizing promutagens to active mutagens. There are a large number of S. cerevisiae strains with varying abilities to produce cytochrome P-450. However, strain selection and ideal cultivation conditions are not well defined. We compared cytochrome P-450 levels in four different S. cerevisiae strains and evaluated the cultivation conditions necessary to obtain the highest levels. The amount of cytochrome P-450 produced by each strain varied, as did the incubation time needed to reach the maximum level. The highest cytochrome P-450 concentrations were found in media containing fermentable sugars. The NCYC 240 strain produced the highest level of cytochrome P-450 when grown in the presence of 20 % (w/v glucose. The addition of ethanol to the media also increased cytochrome P-450 synthesis in this strain. These results indicate cultivation conditions must be specific and well-established for the strain selected in order to assure high cytochrome P-450 levels and reliable mutagenicity results.Linhagens de Saccharomyces cerevisiae tem sido amplamente empregadas em testes de mutagenicidade devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias pró-mutagênicas à sua forma ativa. Devido à grande variedade de linhagens de S. cerevisiae com diferentes capacidades de produção de citocromo P-450, torna-se necessária a seleção de cepas, bem como a definição das condições ideais de cultivo. Neste trabalho, foram comparados os níveis de citocromo P-450 em quatro diferentes linhagens de S. cerevisiae e avaliadas as condições de cultivo necessárias para obtenção de altas concentrações deste sistema enzimático. O maior nível enzimático foi encontrado na linhagem NCYC 240 em presença de 20 % de glicose (p/v. A adição de etanol ao meio de cultura também produziu um aumento na s

Energy transfer dynamics in an RC-LH1-PufX tubular photosynthetic membrane

International Nuclear Information System (INIS)

Hsin, J; Sener, M; Schulten, K; Struempfer, J; Qian, P; Hunter, C N

2010-01-01

Light absorption and the subsequent transfer of excitation energy are the first two steps in the photosynthetic process, carried out by protein-bound pigments, mainly bacteriochlorophylls (BChls), in photosynthetic bacteria. BChls are anchored in light-harvesting (LH) complexes, such as light-harvesting complex I (LH1), which directly associates with the reaction center (RC), forming the RC-LH1 core complex. In Rhodobacter sphaeroides, RC-LH1 core complexes contain an additional protein, PufX, and assemble into dimeric RC-LH1-PufX core complexes. In the absence of LH complex II (LH2), the former complexes can aggregate into a helically ordered tubular photosynthetic membrane. We have examined the excitation transfer dynamics in a single RC-LH1-PufX core complex dimer using the hierarchical equations of motion for dissipative quantum dynamics that accurately, yet in a computationally costly manner, treat the coupling between BChls and their protein environment. A widely employed description, the generalized Foerster (GF) theory, was also used to calculate the transfer rates of the same excitonic system in order to verify the accuracy of this computationally cheap method. Additionally, in light of the structural uncertainties in the Rba. sphaeroides RC-LH1-PufX core complex, geometrical alterations were introduced into the BChl organization. It is shown that the energy transfer dynamics are not affected by the considered changes in the BChl organization and that the GF theory provides accurate transfer rates. An all-atom model for a tubular photosynthetic membrane is then constructed on the basis of electron microscopy data, and the overall energy transfer properties of this membrane are computed.

Energy transfer dynamics in an RC-LH1-PufX tubular photosynthetic membrane

Energy Technology Data Exchange (ETDEWEB)

Hsin, J; Sener, M; Schulten, K [Department of Physics and Beckman Institute, University of Illinois at Urbana-Champaign, Urbana (United States); Struempfer, J [Center for Biophysics and Computational Biology and Beckman Institute, University of Illinois at Urbana-Champaign, Urbana (United States); Qian, P; Hunter, C N, E-mail: kschulte@ks.uiuc.ed [Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN (United Kingdom)

2010-08-15

Light absorption and the subsequent transfer of excitation energy are the first two steps in the photosynthetic process, carried out by protein-bound pigments, mainly bacteriochlorophylls (BChls), in photosynthetic bacteria. BChls are anchored in light-harvesting (LH) complexes, such as light-harvesting complex I (LH1), which directly associates with the reaction center (RC), forming the RC-LH1 core complex. In Rhodobacter sphaeroides, RC-LH1 core complexes contain an additional protein, PufX, and assemble into dimeric RC-LH1-PufX core complexes. In the absence of LH complex II (LH2), the former complexes can aggregate into a helically ordered tubular photosynthetic membrane. We have examined the excitation transfer dynamics in a single RC-LH1-PufX core complex dimer using the hierarchical equations of motion for dissipative quantum dynamics that accurately, yet in a computationally costly manner, treat the coupling between BChls and their protein environment. A widely employed description, the generalized Foerster (GF) theory, was also used to calculate the transfer rates of the same excitonic system in order to verify the accuracy of this computationally cheap method. Additionally, in light of the structural uncertainties in the Rba. sphaeroides RC-LH1-PufX core complex, geometrical alterations were introduced into the BChl organization. It is shown that the energy transfer dynamics are not affected by the considered changes in the BChl organization and that the GF theory provides accurate transfer rates. An all-atom model for a tubular photosynthetic membrane is then constructed on the basis of electron microscopy data, and the overall energy transfer properties of this membrane are computed.

Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c

Energy Technology Data Exchange (ETDEWEB)

Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, I-00146 Roma (Italy); Ciaccio, Chiara [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , I-00133 Roma (Italy); Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, I-70126 Bari (Italy); Sinibaldi, Federica; Santucci, Roberto [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , I-00133 Roma (Italy); Coletta, Massimo [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , I-00133 Roma (Italy); Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, I-70126 Bari (Italy)

2011-01-07

Research highlights: {yields} Cardiolipin binding to cytochrome c. {yields} Cardiolipin-dependent peroxynitrite isomerization by cytochrome c. {yields} Cardiolipin-cytochrome c complex plays pro-apoptotic effects. {yields} Cardiolipin-cytochrome c complex plays anti-apoptotic effects. -- Abstract: Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k{sub on}) is (3.2 {+-} 0.4) x 10{sup 5} M{sup -1} s{sup -1}. The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1 {+-} 0.8) x 10{sup -5} M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.

Oleamide synthesizing activity from rat kidney: identification as cytochrome c.

Science.gov (United States)

Driscoll, William J; Chaturvedi, Shalini; Mueller, Gregory P

2007-08-03

Oleamide (cis-9-octadecenamide) is the prototype member of an emerging class of lipid signaling molecules collectively known as the primary fatty acid amides. Current evidence suggests that oleamide participates in the biochemical mechanisms underlying the drive to sleep, thermoregulation, and antinociception. Despite the potential importance of oleamide in these physiologic processes, the biochemical pathway for its synthesis in vivo has not been established. We report here the discovery of an oleamide synthetase found in rat tissues using [(14)C]oleoyl-CoA and ammonium ion. Hydrogen peroxide was subsequently found to be a required cofactor. The enzyme displayed temperature and pH optima in the physiologic range, a remarkable resistance to proteolysis, and specificity for long-chain acyl-CoA substrates. The reaction demonstrated Michaelis-Menten kinetics with a K(m) for oleoyl-CoA of 21 microm. Proteomic, biochemical, and immunologic analyses were used to identify the source of the oleamide synthesizing activity as cytochrome c. This identification was based upon peptide mass fingerprinting of isolated synthase protein, a tight correlation between enzymatic activity and immunoreactivity for cytochrome c, and identical functional properties shared by the tissue-derived synthetase and commercially obtained cytochrome c. The ability of cytochrome c to catalyze the formation of oleamide experimentally raises the possibility that cytochrome c may mediate oleamide biosynthesis in vivo.

Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

International Nuclear Information System (INIS)

Kresheck, G.C.; Erman, J.E.

1988-01-01

Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (t/sub m/) were 43.9 +- 1.4 and 63.3 +- 1.6 0 C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +- 1.3 0 C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase

The swimming of a perfect deforming helix

Science.gov (United States)

Koens, Lyndon; Zhang, Hang; Mourran, Ahmed; Lauga, Eric

2017-11-01

Many bacteria rotate helical flagellar filaments in order to swim. When at rest or rotated counter-clockwise these flagella are left handed helices but they undergo polymorphic transformations to right-handed helices when the motor is reversed. These helical deformations themselves can generate motion, with for example Rhodobacter sphaeroides using the polymorphic transformation of the flagellum to generate rotation, or Spiroplasma propagating a change of helix handedness across its body's length to generate forward motion. Recent experiments reported on an artificial helical microswimmer generating motion without a propagating change in handedness. Made of a temperature sensitive gel, these swimmers moved by changing the dimensions of the helix in a non-reciprocal way. Inspired by these results and helix's ubiquitous presence in the bacterial world, we investigate how a deforming helix moves within a viscous fluid. Maintaining a single handedness along its entire length, we discuss how a perfect deforming helix can create a non-reciprocal swimming stroke, identify its principle directions of motion, and calculate the swimming kinematics asymptotically.

Detection of Singlet Oxygen Formation inside Photoactive Biohybrid Composite Material

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Kata Hajdu

2017-12-01

Full Text Available Photosynthetic reaction center proteins (RCs are the most efficient light energy converter systems in nature. The first steps of the primary charge separation in photosynthesis take place in these proteins. Due to their unique properties, combining RCs with nano-structures promising applications can be predicted in optoelectronic systems. In the present work RCs purified from Rhodobacter sphaeroides purple bacteria were immobilized on multiwalled carbon nanotubes (CNTs. Carboxyl—and amine-functionalised CNTs were used, so different binding procedures, physical sorption and chemical sorption as well, could be applied as immobilization techniques. Light-induced singlet oxygen production was measured in the prepared photoactive biocomposites in water-based suspension by histidine mediated chemical trapping. Carbon nanotubes were applied under different conditions in order to understand their role in the equilibration of singlet oxygen concentration in the suspension. CNTs acted as effective quenchers of 1O2 either by physical (resonance energy transfer or by chemical (oxidation reaction and their efficiency showed dependence on the diffusion distance of 1O2.

A structural basis for electron transfer in bacterial photosynthesis

International Nuclear Information System (INIS)

Norris, J.R.; DiMagno, T.J.; Angerhofer, A.; Chang, C.H.; El-Kabbani, O.; Schiffer, M.

1989-01-01

Triplet data for the primary donor in single crystals of bacterial reaction centers of Rhodobacter sphaeroides and Rhodopseudomonas viridis are interpreted in terms of the corresponding x-ray structures. The analysis of electron paramagnetic resonance data from single crystals (triplet zero field splitting and cation and triplet linewidth of the primary special pair donor of bacterial reaction centers) is extended to systems of a non-crystalline nature. A unified interpretation based on frontier molecular orbitals concludes that the special pair behaves like a supermolecule in all wild-type bacteria investigated here. However, in heterodimers of Rb. capsulatus (His M200 changed to Leu or Phe with the result that the M-half of the special pair is converted to bacteriopheophytin) the special pair possesses the EPR properties more appropriately described in terms of a monomer. In all cases the triplet state and cation EPR properties appear to be dominated by the highest occupied molecular orbitals. These conclusions derived from EPR experiments are supplemented by data from Stark spectroscopy of reaction centers from Rb. capsulatus. 41 refs., 3 tabs

Hydrogen bonds in the vicinity of the special pair of the bacterial reaction center probed by hydrostatic high-pressure absorption spectroscopy.

Science.gov (United States)

Kangur, Liina; Jones, Michael R; Freiberg, Arvi

2017-12-01

Using the native bacteriochlorophyll a pigment cofactors as local probes, we investigated the response to external hydrostatic high pressure of reaction center membrane protein complexes from the photosynthetic bacterium Rhodobacter sphaeroides. Wild-type and engineered complexes were used with a varied number (0, 1 or 2) of hydrogen bonds that bind the reaction center primary donor bacteriochlorophyll cofactors to the surrounding protein scaffold. A pressure-induced breakage of hydrogen bonds was established for both detergent-purified and membrane-embedded reaction centers, but at rather different pressures: between 0.2 and 0.3GPa and at about 0.55GPa, respectively. The free energy change associated with the rupture of the single hydrogen bond present in wild-type reaction centers was estimated to be equal to 13-14kJ/mol. In the mutant with two symmetrical hydrogen bonds (FM197H) a single cooperative rupture of the two bonds was observed corresponding to an about twice stronger bond, rather than a sequential rupture of two individual bonds. Copyright © 2017 Elsevier B.V. All rights reserved.

P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis

OpenAIRE

Murataliev, Marat B.; Guzov, Victor M.; Walker, F. Ann; Feyereisen, René

2008-01-01

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylatio...

Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine

International Nuclear Information System (INIS)

McCue, Jeffrey M.; Driscoll, William J.; Mueller, Gregory P.

2008-01-01

Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo

NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

Science.gov (United States)

Ding, Xinxin; Wolf, C. Roland; Porter, Todd D.; Pandey, Amit V.; Zhang, Qing-Yu; Gu, Jun; Finn, Robert D.; Ronseaux, Sebastien; McLaughlin, Lesley A.; Henderson, Colin J.; Zou, Ling; Flück, Christa E.

2013-01-01

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

Acrolein, A Reactive Product of Lipid Peroxidation, Induces Oxidative Modification of Cytochrome c

Energy Technology Data Exchange (ETDEWEB)

Kang, Jung Hoon [Cheongju Univ., Cheongju (Korea, Republic of)

2013-11-15

Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). In Alzheimer's brain, ACR was found to be elevated in hippocampus and temporal cortex where oxidative stress is high. In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with ACR. When cytochrome c was incubated with ACR, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in ACR-treated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the ACR-mediated cytochrome c modification and carbonyl compound formation. Our data demonstrate that oxidative damage of cytochrome c by ACR might induce disruption of cyotochrome c structure and iron mishandling as a contributing factor to the pathology of AD.

Acrolein, A Reactive Product of Lipid Peroxidation, Induces Oxidative Modification of Cytochrome c

International Nuclear Information System (INIS)

Kang, Jung Hoon

2013-01-01

Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). In Alzheimer's brain, ACR was found to be elevated in hippocampus and temporal cortex where oxidative stress is high. In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with ACR. When cytochrome c was incubated with ACR, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in ACR-treated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the ACR-mediated cytochrome c modification and carbonyl compound formation. Our data demonstrate that oxidative damage of cytochrome c by ACR might induce disruption of cyotochrome c structure and iron mishandling as a contributing factor to the pathology of AD

Disruption of a hydrogen bond network in human versus spider monkey cytochrome c affects heme crevice stability.

Science.gov (United States)

Goldes, Matthew E; Jeakins-Cooley, Margaret E; McClelland, Levi J; Mou, Tung-Chung; Bowler, Bruce E

2016-05-01

The hypothesis that the recent rapid evolution of primate cytochromes c, which primarily involves residues in the least stable Ω-loop (Ω-loop C, residues 40-57), stabilizes the heme crevice of cytochrome c relative to other mammals, is tested. To accomplish this goal, we have compared the properties of human and spider monkey cytochrome c and a set of four variants produced in the process of converting human cytochrome c into spider monkey cytochrome c. The global stability of all variants has been measured by guanidine hydrochloride denaturation. The stability of the heme crevice has been assessed with the alkaline conformational transition. Structural insight into the effects of the five amino acid substitutions needed to convert human cytochrome c into spider monkey cytochrome c is provided by a 1.15Å resolution structure of spider monkey cytochrome c. The global stability for all variants is near 9.0kcal/mol at 25°C and pH7, which is higher than that observed for other mammalian cytochromes c. The heme crevice stability is more sensitive to the substitutions required to produce spider monkey cytochrome c with decreases of up to 0.5 units in the apparent pKa of the alkaline conformational transition relative to human cytochrome c. The structure of spider monkey cytochrome c indicates that the Y46F substitution destabilizes the heme crevice by disrupting an extensive hydrogen bond network that connects three surface loops including Ω-loop D (residues 70-85), which contains the Met80 heme ligand. Copyright © 2015 Elsevier Inc. All rights reserved.

Improving the hydrogen production capacity of Rhodobacter capsulatus by genetically modifying redox balancing pathways

Energy Technology Data Exchange (ETDEWEB)

Oeztuerk, Yavuz [TUEBITAK Research Institute for Genetic Engineering and Biotechnology, Gebze Kocaeli (Turkey); Goekce, Abdulmecit [Istanbul Technical Univ. (Turkey). Dept. of Molecular Biology and Genetics; Guergan, Muazzez; Yuecel, Meral [Middle East Technical Univ., Ankara (Turkey). Dept. of Biology

2010-07-01

In Rhodobacter capsulatus, balancing the oxidation-reduction potential (redox-balance) is maintained via a number of inter-dependent regulatory mechanisms that enable these organisms to accommodate divergent growth modes. In order to maintain redox homeostasis, this bacterium possesses regulatory mechanisms functioning as electron sinks affecting the oxidation-reduction state of the ubiquinone pool. Under the photoheterotrophic growth conditions with reduced carbon sources, the excess reducing equivalents are primarily consumed via the reduction of CO{sub 2} through the Calvin-Benson-Bassham (CBB) pathway or by the reduction of protons into hydrogen with the use of dinitrogenase enzyme system. In this study, our aim was to develop strategies to funnel the excess reducing equivalents to nitrogenase-dependent hydrogen production by blocking the carbon-fixation pathway. To realize this purpose, CO{sub 2} fixation was blocked by inactivating the Phosphoribulokinase (PRK) of CBB pathway in wild type (MT1131), uptake-hydrogenase (YO3) and cyt cbb{sub 3} oxidase deficient (YO4) strains. The hydrogen production capacity of newly generated strains deficient in the Calvin-Benson-Bassham pathway were analyzed and compared with wild type strains. The results indicated that, the hydrogen production efficiency and capacity of R. capsulatus was further improved by directing the excess reducing equivalents to dinitrogenase-dependent hydrogen production. (orig.)

Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase

Energy Technology Data Exchange (ETDEWEB)

Henry, Rowan M; Caplan, David; Pomes, Regis [Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, M5G 1X8 (Canada); Fadda, Elisa, E-mail: pomes@sickkids.ca [Department of Chemistry, University of Galway (Ireland)

2011-06-15

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay

Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase

International Nuclear Information System (INIS)

Henry, Rowan M; Caplan, David; Pomes, Regis; Fadda, Elisa

2011-01-01

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay

Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase

Science.gov (United States)

Henry, Rowan M.; Caplan, David; Fadda, Elisa; Pomès, Régis

2011-06-01

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay

Multi-heme Cytochromes in Shewanella oneidensis MR-1: Structures, functions and opportunities

Energy Technology Data Exchange (ETDEWEB)

Breuer, Marian; Rosso, Kevin M.; Blumberger, Jochen; Butt, Julea N.

2014-11-05

Multi-heme cytochromes are employed by a range of microorganisms to transport electrons over distances of up to tens of nanometers. Perhaps the most spectacular utilization of these proteins is in the reduction of extracellular solid substrates, including electrodes and insoluble mineral oxides of Fe(III) and Mn(III/IV), by species of Shewanella and Geobacter. However, multi-heme cytochromes are found in numerous and phylogenetically diverse prokaryotes where they participate in electron transfer and redox catalysis that contributes to biogeochemical cycling of N, S and Fe on the global scale. These properties of multi-heme cytochromes have attracted much interest and contributed to advances in bioenergy applications and bioremediation of contaminated soils. Looking forward there are opportunities to engage multi-heme cytochromes for biological photovoltaic cells, microbial electrosynthesis and developing bespoke molecular devices. As a consequence it is timely to review our present understanding of these proteins and we do this here with a focus on the multitude of functionally diverse multi-heme cytochromes in Shewanella oneidensis MR-1. We draw on findings from experimental and computational approaches which ideally complement each other in the study of these systems: computational methods can interpret experimentally determined properties in terms of molecular structure to cast light on the relation between structure and function. We show how this synergy has contributed to our understanding of multi-heme cytochromes and can be expected to continue to do so for greater insight into natural processes and their informed exploitation in biotechnologies.

Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction.

Science.gov (United States)

Tavagnacco, Claudio; Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Borsari, Marco

2011-10-21

Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.

Crystallization and preliminary crystallographic studies of FoxE from Rhodobacter ferrooxidans SW2, an FeII oxidoreductase involved in photoferrotrophy

International Nuclear Information System (INIS)

Pereira, L.; Saraiva, I. H.; Coelho, R.; Newman, D. K.; Louro, R. O.; Frazão, C.

2012-01-01

Crystals of the R. ferrooxidans SW2 iron oxidoreductase FoxE were obtained and the phase problem was solved by Fe SAD at 2.44 Å resolution. FoxE is a protein encoded by the foxEYZ operon of Rhodobacter ferrooxidans SW2 that is involved in Fe II -based anoxygenic photosynthesis (‘photoferrotrophy’). It is thought to reside in the periplasm, where it stimulates light-dependent Fe II oxidation. It contains 259 residues, including two haem c-binding motifs. As no three-dimensional model is available and there is no structure with a similar sequence, crystals of FoxE were produced. They diffracted to 2.44 Å resolution using synchrotron radiation at the Fe edge. The phase problem was solved by SAD using SHELXC/D/E and the experimental maps confirmed the presence of two haems per molecule

In-silico assessment of protein-protein electron transfer. a case study: cytochrome c peroxidase--cytochrome c.

Directory of Open Access Journals (Sweden)

Frank H Wallrapp

Full Text Available The fast development of software and hardware is notably helping in closing the gap between macroscopic and microscopic data. Using a novel theoretical strategy combining molecular dynamics simulations, conformational clustering, ab-initio quantum mechanics and electronic coupling calculations, we show how computational methodologies are mature enough to provide accurate atomistic details into the mechanism of electron transfer (ET processes in complex protein systems, known to be a significant challenge. We performed a quantitative study of the ET between Cytochrome c Peroxidase and its redox partner Cytochrome c. Our results confirm the ET mechanism as hole transfer (HT through residues Ala194, Ala193, Gly192 and Trp191 of CcP. Furthermore, our findings indicate the fine evolution of the enzyme to approach an elevated turnover rate of 5.47 × 10(6 s(-1 for the ET between Cytc and CcP through establishment of a localized bridge state in Trp191.

Production, purification and detergent exchange of isotopically labeled Bacillussubtilis cytochrome b₅₅₈ (SdhC).

Science.gov (United States)

Baureder, Michael; Hederstedt, Lars

2011-11-01

Cytochrome b₅₅₈ of the gram-positive bacterium Bacillussubtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinellasuccinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b₅₅₈. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b₅₅₈ produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome bc1 Complex

DEFF Research Database (Denmark)

Barragan, Angela M.; Crofts, Antony R.; Schulten, Klaus

2015-01-01

for the function of the bc1 complex is the initial redox process that involves a bifurcated electron transfer in which the two electrons from a quinol substrate are passed to different electron acceptors in the bc1 complex. The electron transfer is coupled to proton transfer. The overall mechanism of quinol...... all atom molecular dynamics and quantum chemical calculations to reveal the binding modes of quinol at the Qo-site of the bc1 complex from Rhodobacter capsulatus. The calculations suggest a novel configuration of amino acid residues responsible for quinol binding and support a mechanism for proton...

Monoclonal antibodies to drosophila cytochrome P-450's

International Nuclear Information System (INIS)

Sundseth, S.S.; Kennel, S.J.; Waters, L.C.

1987-01-01

Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins

Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Kärenlampi, S O; Marin, E; Hänninen, O O

1981-02-15

The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.

Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase.

Science.gov (United States)

Balodite, Elina; Strazdina, Inese; Galinina, Nina; McLean, Samantha; Rutkis, Reinis; Poole, Robert K; Kalnenieks, Uldis

2014-09-01

The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc1 complex via cytochrome c552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase. © 2014 The Authors.

Fast prediction of cytochrome P450 mediated drug metabolism

DEFF Research Database (Denmark)

Rydberg, Patrik Åke Anders; Poongavanam, Vasanthanathan; Oostenbrink, Chris

2009-01-01

Cytochrome P450 mediated metabolism of drugs is one of the major determinants of their kinetic profile, and prediction of this metabolism is therefore highly relevant during the drug discovery and development process. A new rule-based method, based on results from density functional theory...... calculations, for predicting activation energies for aliphatic and aromatic oxidations by cytochromes P450 is developed and compared with several other methods. Although the applicability of the method is currently limited to a subset of P450 reactions, these reactions describe more than 90...

Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

OpenAIRE

Kärenlampi, S O; Marin, E; Hänninen, O O

1981-01-01

The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture...

Removal of Bound Triton X-100 from Purified Bovine Heart Cytochrome bc1

OpenAIRE

Varhač, Rastislav; Robinson, Neal C.; Musatov, Andrej

2009-01-01

Cytochrome bc1 isolated from Triton X-100 solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nmol of the enzyme. Purified cytochrome bc1 is fully active; however, protein bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc1 was accomplished by incubation with Bio-Beads SM-2 in presence of sodium cholate. Sodium cholate is critical since it does not interfere with the adsorpt...

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

Energy Technology Data Exchange (ETDEWEB)

Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A., E-mail: Michail.Alterman@fda.hhs.gov

2013-02-15

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

International Nuclear Information System (INIS)

Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A.

2013-01-01

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

Kinetics of two-stage fermentation process for the production of hydrogen

Energy Technology Data Exchange (ETDEWEB)

Nath, Kaushik [Department of Chemical Engineering, G.H. Patel College of Engineering and Technology, Vallabh Vidyanagar 388 120, Gujarat (India); Muthukumar, Manoj; Kumar, Anish; Das, Debabrata [Fermentation Technology Laboratory, Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302 (India)

2008-02-15

Two-stage process described in the present work is a combination of dark and photofermentation in a sequential batch mode. In the first stage glucose is fermented to acetate, CO{sub 2} and H{sub 2} in an anaerobic dark fermentation by Enterobacter cloacae DM11. This is followed by a successive second stage where acetate is converted to H{sub 2} and CO{sub 2} in a photobioreactor by photosynthetic bacteria, Rhodobacter sphaeroides O.U. 001. The yield of hydrogen in the first stage was about 3.31molH{sub 2}(molglucose){sup -1} (approximately 82% of theoretical) and that in the second stage was about 1.5-1.72molH{sub 2}(molaceticacid){sup -1} (approximately 37-43% of theoretical). The overall yield of hydrogen in two-stage process considering glucose as preliminary substrate was found to be higher compared to a single stage process. Monod model, with incorporation of substrate inhibition term, has been used to determine the growth kinetic parameters for the first stage. The values of maximum specific growth rate ({mu} {sub max}) and K{sub s} (saturation constant) were 0.398h{sup -1} and 5.509gl{sup -1}, respectively, using glucose as substrate. The experimental substrate and biomass concentration profiles have good resemblance with those obtained by kinetic model predictions. A model based on logistic equation has been developed to describe the growth of R. sphaeroides O.U 001 in the second stage. Modified Gompertz equation was applied to estimate the hydrogen production potential, rate and lag phase time in a batch process for various initial concentration of glucose, based on the cumulative hydrogen production curves. Both the curve fitting and statistical analysis showed that the equation was suitable to describe the progress of cumulative hydrogen production. (author)

Automated ensemble assembly and validation of microbial genomes

Science.gov (United States)

2014-01-01

Background The continued democratization of DNA sequencing has sparked a new wave of development of genome assembly and assembly validation methods. As individual research labs, rather than centralized centers, begin to sequence the majority of new genomes, it is important to establish best practices for genome assembly. However, recent evaluations such as GAGE and the Assemblathon have concluded that there is no single best approach to genome assembly. Instead, it is preferable to generate multiple assemblies and validate them to determine which is most useful for the desired analysis; this is a labor-intensive process that is often impossible or unfeasible. Results To encourage best practices supported by the community, we present iMetAMOS, an automated ensemble assembly pipeline; iMetAMOS encapsulates the process of running, validating, and selecting a single assembly from multiple assemblies. iMetAMOS packages several leading open-source tools into a single binary that automates parameter selection and execution of multiple assemblers, scores the resulting assemblies based on multiple validation metrics, and annotates the assemblies for genes and contaminants. We demonstrate the utility of the ensemble process on 225 previously unassembled Mycobacterium tuberculosis genomes as well as a Rhodobacter sphaeroides benchmark dataset. On these real data, iMetAMOS reliably produces validated assemblies and identifies potential contamination without user intervention. In addition, intelligent parameter selection produces assemblies of R. sphaeroides comparable to or exceeding the quality of those from the GAGE-B evaluation, affecting the relative ranking of some assemblers. Conclusions Ensemble assembly with iMetAMOS provides users with multiple, validated assemblies for each genome. Although computationally limited to small or mid-sized genomes, this approach is the most effective and reproducible means for generating high-quality assemblies and enables users to

A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification.

Directory of Open Access Journals (Sweden)

Saharnaz Bigdeli

Full Text Available Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated

Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

International Nuclear Information System (INIS)

Stiborová, Marie; Moserová, Michaela; Černá, Věra; Indra, Radek; Dračínský, Martin; Šulc, Miroslav; Henderson, Colin J.; Wolf, C. Roland; Schmeiser, Heinz H.; Phillips, David H.; Frei, Eva; Arlt, Volker M.

2014-01-01

In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b 5 , and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ∼1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b 5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b 5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b 5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b 5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR

Polarography of cytochrome c in ammoniacal buffers containing cobalt ions. The effect of the protein conformation.

Science.gov (United States)

Brabec, V

1985-12-01

Catalytic currents yielded by cytochrome c in ammoniacal buffers containing cobalt ions at a dropping mercury electrode (Brdicka's catalytic currents) were investigated by means of direct current, differential pulse, normal pulse (NP) and phase-selective alternating current polarography. It was found that Brdicka's catalytic current of cytochrome c, (the more negative part of Brdicka's double wave, wave B) is influenced by the presence of cytochrome c denaturants in the background solution. The wave B rose with the increasing concentrations of urea and sodium perchlorate, and increased in parallel with absorbance changes at 409 and 695 nm measured for identical cytochrome c solutions. The latter absorbance changes reflect unfolding of cytochrome c molecules in the bulk of solution by these denaturants. The results of NP polarography (a technique working with large potential excursion during the drop lifetime) indicate that in Brdicka's solution cytochrome c could extensively be unfolded due to its adsorption at the mercury electrode, polarized to potentials around that of zero charge.

Production of bioplastics and hydrogen gas by photosynthetic microorganisms

Science.gov (United States)

Yasuo, Asada; Masato, Miyake; Jun, Miyake

1998-03-01

Our efforts have been aimed at the technological basis of photosynthetic-microbial production of materials and an energy carrier. We report here accumulation of poly-(3-hydroxybutyrate) (PHB), a raw material of biodegradable plastics and for production of hydrogen gas, and a renewable energy carrier by photosynthetic microorganisms (tentatively defined as cyanobacteria plus photosynthetic bateria, in this report). A thermophilic cyanobacterium, Synechococcus sp. MA19 that accumulates PHB at more than 20% of cell dry wt under nitrogen-starved conditions was isolated and microbiologically identified. The mechanism of PHB accumulation was studied. A mesophilic Synechococcus PCC7942 was transformed with the genes encoding PHB-synthesizing enzymes from Alcaligenes eutrophus. The transformant accumulated PHB under nitrogen-starved conditions. The optimal conditions for PHB accumulation by a photosynthetic bacterium grown on acetate were studied. Hydrogen production by photosynthetic microorganisms was studied. Cyanobacteria can produce hydrogen gas by nitrogenase or hydrogenase. Hydrogen production mediated by native hydrogenase in cyanobacteria was revealed to be in the dark anaerobic degradation of intracellular glycogen. A new system for light-dependent hydrogen production was targeted. In vitro and in vivo coupling of cyanobacterial ferredoxin with a heterologous hydrogenase was shown to produce hydrogen under light conditions. A trial for genetic trasformation of Synechococcus PCC7942 with the hydrogenase gene from Clostridium pasteurianum is going on. The strong hydrogen producers among photosynthetic bacteria were isolated and characterized. Co-culture of Rhodobacter and Clostriumdium was applied to produce hydrogen from glucose. Conversely in the case of cyanobacteria, genetic regulation of photosynthetic proteins was intended to improve conversion efficiency in hydrogen production by the photosynthetic bacterium, Rhodobacter sphaeroides RV. A mutant acquired by

Conjugation of cytochrome c with hydrogen titanate nanotubes: novel conformational state with implications for apoptosis

Energy Technology Data Exchange (ETDEWEB)

Ray, Moumita; Mazumdar, Shyamalava [Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005 (India); Chatterjee, Sriparna; Das, Tanmay; Bhattacharyya, Somnath; Ayyub, Pushan, E-mail: somnath@tifr.res.in, E-mail: pushan@tifr.res.in, E-mail: shyamal@tifr.res.in [Department of Condensed Matter Physics and Materials Science, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005 (India)

2011-10-14

We show that hydrogen titanate (H{sub 2}Ti{sub 3}O{sub 7}) nanotubes form strongly associated reversible nano-bio-conjugates with the vital respiratory protein, cytochrome c. Resonance Raman spectroscopy along with direct electrochemical studies indicate that in this nano-bio-conjugate, cytochrome c exists in an equilibrium of two conformational states with distinctly different formal redox potentials and coordination geometries of the heme center. The nanotube-conjugated cytochrome c also showed enhanced peroxidase activity similar to the membrane-bound protein that is believed to be an apoptosis initiator. This suggests that such a nanotube-cytochrome c conjugate may be a good candidate for cancer therapy applications.

Evidence that Na+-pumping occurs through the D-channel in Vitreoscilla cytochrome bo

International Nuclear Information System (INIS)

Kim, Seong K.; Stark, Benjamin C.; Webster, Dale A.

2005-01-01

The operon (cyo) encoding the Na + -pumping respiratory terminal oxidase (cytochrome bo) of the bacterium Vitreoscilla was transformed into Escherichia coli GV100, a deletion mutant of cytochrome bo. This was done for the wild type operon and five mutants in three conserved Cyo subunit I amino acids known to be crucial for H + transport in the E. coli enzyme, one near the nuclear center, one in the K-channel, and one in the D-channel. CO-binding, NADH and ubiquinol oxidase, and Na + -pumping activities were all substantially inhibited by each mutation. The wild type Vitreoscilla cytochrome bo can pump Na + against a concentration gradient, resulting in a transmembrane concentration differential of 2-3 orders of magnitude. It is proposed that Vitreoscilla cytochrome bo pumps four Na + through the D-channel to the exterior and transports four H + through the K-channel for the reduction of each O 2

Coordinate regulation of cytochrome and alternative pathway respiration in tobacco.

Science.gov (United States)

Vanlerberghe, G C; McIntosh, L

1992-12-01

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.

Unique organizational and functional features of the cytochrome c maturation system in Shewanella oneidensis.

Directory of Open Access Journals (Sweden)

Miao Jin

Full Text Available Shewanella are renowned for their ability to respire on a wide range of electron acceptors, which has been partially accredited to the presence of a large number of the c-type cytochromes. In the model species S. oneidensis MR-1, at least 41 genes encode c-type cytochromes that are predicted to be intact, thereby likely functional. Previously, in-frame deletion mutants for 36 of these genes were obtained and characterized. In this study, first we completed the construction of an entire set of c-type cytochrome mutants utilizing a newly developed att-based mutagenesis approach, which is more effective and efficient than the approach used previously by circumventing the conventional cloning. Second, we investigated the cytochrome c maturation (Ccm system in S. oneidensis. There are two loci predicted to encode components of the Ccm system, SO0259-SO0269 and SO0476-SO0478. The former is proven essential for cytochrome c maturation whereas the latter is dispensable. Unlike the single operon organization observed in other γ-proteobacteria, genes at the SO0259-SO0269 locus are uniquely organized into four operons, ccmABCDE, scyA, SO0265, and ccmFGH-SO0269. Functional analysis revealed that the SO0265 gene rather than the scyA and SO0269 genes are relevant to cytochrome c maturation.

Cytochrome c interaction with hematite ({alpha}-Fe{sub 2}O{sub 3}) surfaces

Energy Technology Data Exchange (ETDEWEB)

Eggleston, Carrick M. [Department of Geology and Geophysics, University of Wyoming, Laramie, WY 82071 (United States)]. E-mail: carrick@uwyo.edu; Khare, Nidhi [Department of Geology and Geophysics, University of Wyoming, Laramie, WY 82071 (United States); Lovelace, David M. [Department of Geology and Geophysics, University of Wyoming, Laramie, WY 82071 (United States)

2006-02-15

The interaction of metalloproteins such as cytochromes with oxides is of interest for a number of reasons, including molecular catalysis of environmentally important mineral-solution electron transfer reactions (e.g., dehalogenations) and photovoltaic applications. Iron reduction by bacteria, thought to be cytochrome mediated, is of interest for geochemical and environmental remediation reasons. As a baseline for understanding cytochrome interaction with ferric oxide surfaces, we report on the interaction of mitochondrial cytochrome c (Mcc), a well-studied protein, with hematite ({alpha}-Fe{sub 2}O{sub 3}) surfaces. Mcc sorbs strongly to hematite from aqueous solution in a narrow pH range corresponding to opposite charge on Mcc and hematite (between pH 8.5 and 10, Mcc is positively charged and hematite surfaces are negatively charged). Cyclic voltammetry of Mcc using hematite electrodes gives redox potentials characteristic of Mcc in a native conformational state, with no evidence for unfolding on the hematite surface. Atomic force microscopy imaging is consistent with a loosely attached adsorbate that is easily deformed by the AFM tip. In phosphate-containing solution, Mcc adhers to the surface more strongly. These results establish hematite as a viable material for electrochemical and spectroscopic characterization of cytochrome-mineral interaction.

Parameterization of the prosthetic redox centers of the bacterial cytochrome bc(1) complex for atomistic molecular dynamics simulations

DEFF Research Database (Denmark)

Kaszuba, K.; Postila, P. A.; Cramariuc, O.

2013-01-01

studied in large-scale classical molecular dynamics (MD) simulations. In part, this is due to lack of suitable force field parameters, centered atomic point charges in particular, for the complex's prosthetic redox centers. Accurate redox center charges are needed to depict realistically the inter-molecular...... interactions at different redox stages of the cyt bc(1) complex. Accordingly, here we present high-precision atomic point charges for the metal centers of the cyt bc(1) complex of Rhodobacter capsulatus derived from extensive density functional theory calculations, fitted using the restrained electrostatic...

PucC and LhaA direct efficient assembly of the light-harvesting complexes in Rhodobacter sphaeroides

DEFF Research Database (Denmark)

Mothersole, David; Jackson, Philip J.; Vasilev, Cvetelin

2016-01-01

. Here we investigate the assembly of light-harvesting LH2 and reaction centre-light-harvesting1-PufX (RC-LH1-PufX) photosystem complexes using spectroscopy, pull-downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lha......A and pucC mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in ΔlhaA mutants assemble to form normal RC-LH1-PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative...

In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.

Science.gov (United States)

Pearson, Josh T; Siu, Sophia; Meininger, David P; Wienkers, Larry C; Rock, Dan A

2010-03-30

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).

Effect of light intensity and initial pH during hydrogen production by an integrated dark and photofermentation process

Energy Technology Data Exchange (ETDEWEB)

Nath, Kaushik [Department of Chemical Engineering, GH Patel College of Engineering and Technology, Vallabh Vidyanagar 388 120, Gujarat (India); Das, Debabrata [Fermentation Technology Laboratory, Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302 (India)

2009-09-15

Photofermentation was carried out with the spent fermentation broth obtained from the anaerobic dark fermentation in a two-stage process. For the first stage, i.e. dark fermentation Enterobacter cloacae DM 11 was used as hydrogen producing microorganism. For photofermentation Rhodobacter sphaeroides O.U. 001, a photo-heterotrophic purple non-sulfur bacterium, was used. pH study revealed that cumulative hydrogen production was maximum at initial medium pH of 7.0 {+-} 0.2. Biomass yield was also high at the vicinity of pH 7.0 and it decreased as the pH increased from 7.0 to 8.0. Increased light intensity resulted in an increase in the total volume of hydrogen evolved and also hydrogen production rate. However, light conversion efficiency decreased by increasing light intensity. A four-fold increase in light intensity resulted in a three-fold decrease in light conversion efficiency although the cumulative volume of hydrogen gas production increased. It was observed that only a maximum of 0.51% light conversion efficiency could be achieved but at the expense of very low light intensity of 2500 lux (3.75 W m{sup -2}). (author)

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

Science.gov (United States)

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-07-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

Reduction of U(VI) and Toxic Metals by Desulfovibrio Cytochrome C3

Energy Technology Data Exchange (ETDEWEB)

Wall, Judy D

2013-04-11

The central objective of our proposed research was twofold: 1) to investigate the structure-function relationship of Desulfovibrio desulfuricans (now Desulfovibrio alaskensis G20) cytochrome c3 with uranium and 2) to elucidate the mechanism for uranium reduction in vitro and in vivo. Physiological analysis of a mutant of D. desulfuricans with a mutation of the gene encoding the type 1 tetraheme cytochrome c3 had demonstrated that uranium reduction was negatively impacted while sulfate reduction was not if lactate were the electron donor. This was thought to be due to the presence of a branched pathway of electron flow from lactate leading to sulfate reduction. Our experimental plan was to elucidate the structural and mechanistic details of uranium reduction involving cytochrome c3.

Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.

Science.gov (United States)

Sacco, James C; Trepanier, Lauren A

2010-01-01

NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (Phydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

Dynamic movement of cytochrome c from mitochondria into cytosol and peripheral circulation in massive hepatic cell injury.

Science.gov (United States)

Kobayashi, Yoshinori; Mori, Masaaki; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Imagawa, Tomoyuki; Yokota, Shumpei

2004-12-01

In the process of apoptosis, it is known that the transition of cytochrome c from mitochondria into the cytosol occurs, and tumor necrosis factor (TNF)-alpha is one of the molecules responsible for this event. But in the state of hypercytokine induced by D-galactosamine (D-GaIN)/Lipopolysaccharide (LPS), the localization of cytochrome c is little known. Rats were administrated with D-GaIN(700 mg/kg)/LPS(200 microg/kg). Blood and tissue samples were collected and examined for levels of pro-inflammatory cytokines, the apoptosis of liver cells, and the localization of cytochrome c. Before administration of D-GaIN/LPS, cytochrome c was definitely localized in the mitochondria. At 2 h after simultaneous administration of D-GaIN/LPS, cytochrome c had accumulated in the cytosol following abrupt increases of plasma TNF-alpha. Massive cell destruction due to apoptosis proved by Terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling staining was observed in liver tissue 4 h later and markedly increased levels of cytochrome c were detected in the plasma 12 h after D-GaIN/LPS administration. Liver injury induced by simultaneous administration of D-GaIN/LPS was closely associated with the production of TNF-alpha, and also with the dynamic movement of cytochrome c from the mitochondria into the cytosol, and then into the systemic circulation. The detection of plasma cytochrome c levels may be a useful clinical tool for the detection of apoptosis in vivo.

Formation of putative chloroplast cytochromes in isolated developing pea chloroplasts

International Nuclear Information System (INIS)

Thaver, S.S.; Bhava, D.; Castelfranco, P.A.

1986-01-01

In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea chloroplasts were incubated with [ 14 C]-5-aminolevulinic acid [ 14 C]-ALA. The major labeled band (M/sub r/ = 43 kDa by LDS-PAGE) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H 2 O 2 stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [ 14 C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome. The effect of exogenous iron, iron chelators, gabaculine (an inhibitor of ALA synthesis) and other incubation conditions upon the in vitro formation of putative chloroplast cytochromes will be discussed

Cytochrome c6B of Synechococcus sp. WH 8102 – Crystal structure and basic properties of novel c6-like family representative

International Nuclear Information System (INIS)

Zatwarnicki, Pawel; Barciszewski, Jakub; Krzywda, Szymon; Jaskolski, Mariusz; Kolesinski, Piotr; Szczepaniak, Andrzej

2014-01-01

Highlights: • Crystal structure of cytochrome c 6B from Synechococcus sp. WH 8102 was solved. • Basic biophysical properties of cytochrome c 6B were determined. • Cytochrome c 6B exhibits similar architecture to cytochrome c 6 . • Organization of heme binding pocket of cytochrome c 6B differs from that of c 6 . • Midpoint potential of cytochrome c 6B is significantly lower than of cytochrome c 6 . - Abstract: Cytochromes c are soluble electron carriers of relatively low molecular weight, containing single heme moiety. In cyanobacteria cytochrome c 6 participates in electron transfer from cytochrome b 6 f complex to photosystem I. Recent phylogenetic analysis revealed the existence of a few families of proteins homologous to the previously mentioned. Cytochrome c 6A from Arabidopsis thaliana was identified as a protein responsible for disulfide bond formation in response to intracellular redox state changes and c 550 is well known element of photosystem II. However, function of cytochromes marked as c 6B , c 6C and c M as well as the physiological process in which they take a part still remain unidentified. Here we present the first structural and biophysical analysis of cytochrome from the c 6B family from mesophilic cyanobacteria Synechococcus sp. WH 8102. Purified protein was crystallized and its structure was refined at 1.4 Å resolution. Overall architecture of this polypeptide resembles typical I-class cytochromes c. The main features, that distinguish described protein from cytochrome c 6 , are slightly red-shifted α band of UV–Vis spectrum as well as relatively low midpoint potential (113.2 ± 2.2 mV). Although, physiological function of cytochrome c 6B has yet to be determined its properties probably exclude the participation of this protein in electron trafficking between b 6 f complex and photosystem I

Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency

International Nuclear Information System (INIS)

Meier, U.T.; Meyer, U.A.

1987-01-01

The metabolism of the anticonvulsant mephenytoin is subject to a genetic polymorphism. In 2-5% of Caucasians and 18-23% of Japanese subjects a specific cytochrome P-450 isozyme, P-450 meph, is functionally deficient or missing. The authors have accumulated evidence that autoimmune antibodies observed in sera of patients with tienilic acid induced hepatitis (anti-liver kidney microsome 2 or anti-LKM2 antibodies) specifically recognize the cytochrome P-450 involved in the mephrenytoin hydroxylation polymorphism. This is demonstrated by immunoinhibition and immunoprecipitation of microsomal (S)-mephenytoin 4-hydroxylation activity and by the recognition by anti-LKM2 antibodies of a single [ 125 I]-protein band on immunoblots of human liver microsomes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. The cytochrome P-450 recognized by anti-LKM2 antibodies was immunopurified from microsomes derived from livers of extensive (EM) or poor metabolizers (PM) of (S)-mephenytoin. Comparison of the EM-type cytochrome P-450 to that isolated from PM livers revealed no difference in regard to immuno-cross-reactivity, molecular weight, isoelectric point, relative content in microsomes, two-dimensional tryptic peptide maps, one-dimensional peptide maps with three proteases, amino acid composition, and amino-terminal protein sequence. Finally, the same protein was precipitated from microsomes prepared from the liver biopsy of a subject phenotyped in vivo as a poor metabolizer of mephenytoin. These data strongly suggest that the mephenytoin hydroxylation deficiency is caused by a minor structural change leading to a functionally altered cytochrome P-450 isozyme

North African genetic variation of cytochrome and sulfotransferase ...

African Journals Online (AJOL)

in these genes have shown relevant ethnic differences among Sub-Saharan .... This cytochrome catalyzes a big amount of oxidative reactions of substances like ... with samples of European and African origin (because of the scarce data ...

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

Directory of Open Access Journals (Sweden)

Shilpi Khare

2015-07-01

Full Text Available Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1 in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50 of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

Tributyltin interacts with mitochondria and induces cytochrome c release.

Science.gov (United States)

Nishikimi, A; Kira, Y; Kasahara, E; Sato, E F; Kanno, T; Utsumi, K; Inoue, M

2001-01-01

Although triorganotins are potent inducers of apoptosis in various cell types, the critical targets of these compounds and the mechanisms by which they lead to cell death remain to be elucidated. There are two major pathways by which apoptotic cell death occurs: one is triggered by a cytokine mediator and the other is by a mitochondrion-dependent mechanism. To elucidate the mechanism of triorganotin-induced apoptosis, we studied the effect of tributyltin on mitochondrial function. We found that moderately low doses of tributyltin decrease mitochondrial membrane potential and induce cytochrome c release by a mechanism inhibited by cyclosporine A and bongkrekic acid. Tributyltin-induced cytochrome c release is also prevented by dithiols such as dithiothreitol and 2,3-dimercaptopropanol but not by monothiols such as GSH, N-acetyl-L-cysteine, L-cysteine and 2-mercaptoethanol. Further studies with phenylarsine oxide agarose revealed that tributyltin interacts with the adenine nucleotide translocator, a functional constituent of the mitochondrial permeability transition pore, which is selectively inhibited by dithiothreitol. These results suggest that, at low doses, tributyltin interacts selectively with critical thiol residues in the adenine nucleotide translocator and opens the permeability transition pore, thereby decreasing membrane potential and releasing cytochrome c from mitochondria, a series of events consistent with established mechanistic models of apoptosis. PMID:11368793

Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450

International Nuclear Information System (INIS)

Marinello, A.J.; Bansal, S.K.; Paul, B.; Koser, P.L.; Love, J.; Struck, R.F.; Gurtoo, H.L.

1984-01-01

The hepatic cytochrome P-450-mediated metabolism and metabolic activation of [chloroethyl-3H]cyclophosphamide [( chloroethyl-3H]CP) and [4-14C]cyclophosphamide [( 4-14C]CP) were investigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of [14C]acrolein, a metabolite of [4-14C]CP, were also investigated. The metabolism of [chloroethyl-3H]CP and [4-14C]CP to polar metabolites was found to depend on the presence of NADPH and showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reductase. Km and Vmax values were essentially similar. The patterns of inhibition by microsomal mixed-function oxidase inhibitors, anti-cytochrome P-450 antibody, and heat denaturation of the cytochrome P-450 were essentially similar, with subtle differences between [4-14C]CP and [chloroethyl-3H]CP metabolism. The in vitro metabolic activation of CP in the reconstituted system demonstrated predominant binding of [chloroethyl-3H]CP to nucleic acids and almost exclusive binding of [4-14C]CP to proteins. Gel electrophoresis-fluorography of the proteins in the reconstituted system treated with [4-14C]CP demonstrated localization of the 14C label in the cytochrome P-450 region. To examine this association further, hepatic microsomes were modified with [14C]acrolein in the presence and the absence of NADPH. The results confirmed covalent association between [14C]acrolein and cytochrome P-450 in the microsomes and also demonstrated further metabolism of [14C]acrolein, apparently to an epoxide, which is capable of binding covalently to proteins. The results of these investigations not only confirm the significance of primary metabolism but also emphasize the potential role of the secondary metabolism of cyclophosphamide in some of its toxic manifestations

Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

Science.gov (United States)

Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

1988-11-01

In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative.

Genetic defects of cytochrome c oxidase assembly

Czech Academy of Sciences Publication Activity Database

Pecina, Petr; Houšťková, H.; Hansíková, H.; Zeman, J.; Houštěk, Josef

2004-01-01

Roč. 53, Suppl. 1 (2004), s. S213-S223 ISSN 0862-8408 R&D Projects: GA ČR GA303/03/0749 Institutional research plan: CEZ:AV0Z5011922 Keywords : cytochrome c oxidase * mitochondrial disorders Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.140, year: 2004

Interface Adsorption Taking the Most Advantageous Conformation for Electron Transfer Between Graphene and Cytochrome c.

Science.gov (United States)

Hu, Benfeng; Ge, Zhenpeng; Li, Xiaoyi

2015-07-01

Most designed functions in biomedical nanotechnology are directly influenced by interactions of biological molecules with nano surfaces. Here, we explored and detected the most favorable adsorption conformation of cytochrome c on graphene by measuring the adsorption energy, the number of contact atoms, and the minimal distance between protein and surface. From the root mean square deviation of the protein backbone, the radius of gyration, and the proportion of secondary structure, it is revealed that cytochrome c does not deform significantly and the secondary structures are preserved to a large extent. The residues, Lys, Phe and Thr contribute significantly to the adsorption of cytochrome c to graphene. The long hydrophobic and flexible alkyl tail of Lys, the π-π stacking interaction between Phe and graphene, and the presence of abundant Thr constitute the driving force for the stable adsorption of cytochrome c on graphene. Cytochrome c is adsorbed to graphene with the group heme lying almost perpendicular to the graphene, and the distance between Fe atom and the graphene is 10.15 A, which is shorter than that between electron donor and receptor in many other biosystems. All the results suggest that the most favorable adsorption takes the most advantageous conformation for electron transfer, which promotes significantly the electron transfer between graphene and cytochrome c. The findings might provide new and important information for designs of biomedical devices or products with graphene-based nanomaterials.

Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b

DEFF Research Database (Denmark)

Grimmelikhuijzen, C J; Marres, C A; Slater, Conor

1975-01-01

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reo...

Isolation and purification of membrane-bound cytochrome c from ...

African Journals Online (AJOL)

Administrator

2007-05-02

ferrochrome and redox spectra showed the presence of heme-c. Key words: Cytochrome c, respiratory chain and Proteus mirabilis. INTRODUCTION. Proteus mirabilis is facultative anaerobic, rod-shaped, gram negative bacterium.

Effects of Muscle-Specific Oxidative Stress on Cytochrome c Release and Oxidation-Reduction Potential Properties.

Science.gov (United States)

Ke, Yiling; Mitacek, Rachel M; Abraham, Anupam; Mafi, Gretchen G; VanOverbeke, Deborah L; DeSilva, Udaya; Ramanathan, Ranjith

2017-09-06

Mitochondria play a significant role in beef color. However, the role of oxidative stress in cytochrome c release and mitochondrial degradation is not clear. The objective was to determine the effects of display time on cytochrome c content and oxidation-reduction potential (ORP) of beef longissimus lumborum (LL) and psoas major (PM) muscles. PM discolored by day 3 compared with LL. On day 0, mitochondrial content and mitochondrial oxygen consumption were greater in PM than LL. However, mitochondrial content and oxygen consumption were lower (P stress can affect cytochrome c release and ORP changes.

Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

OpenAIRE

Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

2012-01-01

There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. O...

The functional localization of cytochromes b in the respiratory chain of anaerobically grown Proteus mirabilis

NARCIS (Netherlands)

Van Wielink, J E; Reijnders, W N; Van Spanning, R J; Oltmann, L F; Stouthamer, A.H.

1986-01-01

The functional localization of the cytochromes b found in anaerobically grown Proteus mirabilis was investigated. From light absorption spectra, scanned during uninhibited and HQNO-inhibited electron transport to various electron acceptors, it was concluded that all cytochromes b function between

The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA.

Science.gov (United States)

Kuchinski, Kevin S; Brimacombe, Cedric A; Westbye, Alexander B; Ding, Hao; Beatty, J Thomas

2016-02-01

The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ∼4-kb fragments of the producing cell genome, and are released from a subpopulation (SOS response in many bacteria, as a regulator of RcGTA activity. Deletion of the lexA gene resulted in the abolition of detectable RcGTA production and an ∼10-fold reduction in recipient capability. A search for SOS box sequences in the R. capsulatus genome sequence identified a number of putative binding sites located 5' of typical SOS response coding sequences and also 5' of the RcGTA regulatory gene cckA, which encodes a hybrid histidine kinase homolog. Expression of cckA was increased >5-fold in the lexA mutant, and a lexA cckA double mutant was found to have the same phenotype as a ΔcckA single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the lexA mutant is largely due to the overexpression of cckA. This work describes an unusual phenotype of a lexA mutant of the alphaproteobacterium Rhodobacter capsulatus in respect to the phage transduction-like genetic exchange carried out by the R. capsulatus gene transfer agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this lexA mutation not only abolishes the production of RcGTA particles but also impairs the ability of cells to receive RcGTA-borne genes. The data show that, despite an apparent evolutionary relationship to lambdoid phages, the regulation of RcGTA gene expression differs radically. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

Science.gov (United States)

Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

1999-01-01

Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

Early Decrease in Respiration and Uncoupling Event Independent of Cytochrome c Release in PC12 Cells Undergoing Apoptosis

Science.gov (United States)

Berghella, Libera; Ferraro, Elisabetta

2012-01-01

Cytochrome c is a key molecule in mitochondria-mediated apoptosis. It also plays a pivotal role in cell respiration. The switch between these two functions occurs at the moment of its release from mitochondria. This process is therefore extremely relevant for the fate of the cell. Since cytochrome c mediates respiration, we studied the changes in respiratory chain activity during the early stages of apoptosis in order to contribute to unravel the mechanisms of cytochrome c release. We found that, during staurosporine (STS)- induced apoptosis in PC12 cells, respiration is affected before the release of cytochrome c, as shown by a decrease in the endogenous uncoupled respiration and an uncoupling event, both occurring independently of cytochrome c release. The decline in the uncoupled respiration occurs also upon Bcl-2 overexpression (which inhibits cytochrome c release), while the uncoupling event is inhibited by Bcl-2. We also observed that the first stage of nuclear condensation during STS-induced apoptosis does not depend on the release of cytochrome c into the cytosol and is a reversibile event. These findings may contribute to understand the mechanisms affecting mitochondria during the early stages of apoptosis and priming them for the release of apoptogenic factors. PMID:22666257

Modeling of interaction between cytochrome c and the WD domains of Apaf-1: bifurcated salt bridges underlying apoptosome assembly.

Science.gov (United States)

Shalaeva, Daria N; Dibrova, Daria V; Galperin, Michael Y; Mulkidjanian, Armen Y

2015-05-27

Binding of cytochrome c, released from the damaged mitochondria, to the apoptotic protease activating factor 1 (Apaf-1) is a key event in the apoptotic signaling cascade. The binding triggers a major domain rearrangement in Apaf-1, which leads to oligomerization of Apaf-1/cytochrome c complexes into an apoptosome. Despite the availability of crystal structures of cytochrome c and Apaf-1 and cryo-electron microscopy models of the entire apoptosome, the binding mode of cytochrome c to Apaf-1, as well as the nature of the amino acid residues of Apaf-1 involved remain obscure. We investigated the interaction between cytochrome c and Apaf-1 by combining several modeling approaches. We have applied protein-protein docking and energy minimization, evaluated the resulting models of the Apaf-1/cytochrome c complex, and carried out a further analysis by means of molecular dynamics simulations. We ended up with a single model structure where all the lysine residues of cytochrome c that are known as functionally-relevant were involved in forming salt bridges with acidic residues of Apaf-1. This model has revealed three distinctive bifurcated salt bridges, each involving a single lysine residue of cytochrome c and two neighboring acidic resides of Apaf-1. Salt bridge-forming amino acids of Apaf-1 showed a clear evolutionary pattern within Metazoa, with pairs of acidic residues of Apaf-1, involved in bifurcated salt bridges, reaching their highest numbers in the sequences of vertebrates, in which the cytochrome c-mediated mechanism of apoptosome formation seems to be typical. The reported model of an Apaf-1/cytochrome c complex provides insights in the nature of protein-protein interactions which are hard to observe in crystallographic or electron microscopy studies. Bifurcated salt bridges can be expected to be stronger than simple salt bridges, and their formation might promote the conformational change of Apaf-1, leading to the formation of an apoptosome. Combination of

Oxygen and xenobiotic reductase activities of cytochrome P450.

NARCIS (Netherlands)

Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

1995-01-01

The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

Ubiquinol-cytochrome c reductase (Complex III) electrochemistry at multi-walled carbon nanotubes/Nafion modified glassy carbon electrodes

International Nuclear Information System (INIS)

Pelster, Lindsey N.; Minteer, Shelley D.

2012-01-01

Highlights: ► The electron transport chain is important to the understanding of metabolism in the living cell. ► Ubiquinol-cytochrome c reductase is a membrane bound complex of the electron transport chain (Complex III). ► The paper details the first bioelectrochemical characterization of ubiquinol-cytochrome c reductase at an electrode. - Abstract: Electron transport chain complexes are critical to metabolism in living cells. Ubiquinol-cytochrome c reductase (Complex III) is responsible for carrying electrons from ubiquinol to cytochrome c, but the complex has not been evaluated electrochemically. This work details the bioelectrochemistry of ubiquinol-cytochrome c reductase of the electron transport chain of tuber mitochondria. The characterization of the electrochemistry of this enzyme is investigated in carboxylated multi-walled carbon nanotube/tetrabutyl ammonium bromide-modified Nafion ® modified glassy carbon electrodes by cyclic voltammetry. Increasing concentrations of cytochrome c result in a catalytic response from the active enzyme in the nanotube sandwich. The experiments show that the enzyme followed Michaelis–Menten kinetics with a K m for the immobilized enzyme of 2.97 (±0.11) × 10 −6 M and a V max of 6.31 (±0.82) × 10 −3 μmol min −1 at the electrode, but the K m and V max values decreased compared to the free enzyme in solution, which is expected for immobilized redox proteins. This is the first evidence of ubiquinol-cytochrome c reductase bioelectrocatalysis.

[Immunomodulators with an 8-azasteroid structure as inducers of liver cytochrome P-450].

Science.gov (United States)

Kuz'mitskiĭ, B B; Dad'kov, I G; Mashkovich, A E; Stoma, O V; Slepneva, L M

1990-01-01

Two structural analogues of D-homo-8-azasteroids, both an immunostimulant and an immunodepressant, are inductors of the liver cytochrome P-450 in animals. This capability was shown by means of both a decrease of the hexenal sleep duration in the pharmacological test and an increase of the quantity of cytochrome P-450 and the rate of N-demethylation of aminopyrine in the biochemical assays.

The anti-mycobacterial activity of the cytochrome bcc inhibitor Q203 can be enhanced by small-molecule inhibition of cytochrome bd.

NARCIS (Netherlands)

Lu, P.; Asseri, A.H.O.; Kremer, Martijn; Maaskant, Janneke; Ummels, Roy; Lill, H.; Bald, D.

2018-01-01

Mycobacterial energy metabolism currently attracts strong attention as new target space for development of anti-tuberculosis drugs. The imidazopyridine Q203 targets the cytochrome bcc complex of the respiratory chain, a key component in energy metabolism. Q203 blocks growth of Mycobacterium

Rhodobacter capsulatus gains a competitive advantage from respiratory nitrate reduction during light-dark transitions.

Science.gov (United States)

Ellington, M J K; Richardson, D J; Ferguson, S J

2003-04-01

Rhodobacter capsulatus N22DNAR(+) possesses a periplasmic nitrate reductase and is capable of reducing nitrate to nitrite under anaerobic conditions. In the absence of light this ability cannot support chemoheterotrophic growth in batch cultures. This study investigated the effect of nitrate reduction on the growth of R. capsulatus N22DNAR(+) during multiple light-dark cycles of anaerobic photoheterotrophic/dark chemoheterotrophic growth conditions in carbon-limited continuous cultures. The reduction of nitrate did not affect the photoheterotrophic growth yield of R. capsulatus N22DNAR(+). After a transition from photoheterotrophic to dark chemoheterotrophic growth conditions, the reduction of nitrate slowed the initial washout of a R. capsulatus N22DNAR(+) culture. Towards the end of a period of darkness nitrate-reducing cultures maintained higher viable cell counts than non-nitrate-reducing cultures. During light-dark cycling of a mixed culture, the strain able to reduce nitrate (N22DNAR(+)) outcompeted the strain which was unable to reduce nitrate (N22). The evidence indicates that the periplasmic nitrate reductase activity supports slow growth that retards the washout of a culture during anaerobic chemoheterotrophic conditions, and provides a protonmotive force for cell maintenance during the dark period before reillumination. This translates into a selective advantage during repeated light-dark cycles, such that in mixed culture N22DNAR(+) outcompetes N22. Exposure to light-dark cycles will be a common feature for R. capsulatus in its natural habitats, and this study shows that nitrate respiration may provide a selective advantage under such conditions.

The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance.

Directory of Open Access Journals (Sweden)

Julia Leclerc

Full Text Available Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance.

IDENTIFIKASI DAGING BABI MENGGUNAKAN METODE PCR-RFLP GEN Cytochrome b DAN PCR PRIMER SPESIFIK GEN AMELOGENIN (Pork Identification Using PCR-RFLP of Cytochrome b Gene and Species Specific PCR of Amelogenin Gene

Directory of Open Access Journals (Sweden)

Yuny Erwanto

2013-03-01

Full Text Available A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP and species specific PCR methods had been applied for identifying pork in mixture of meat. Pork sample in various levels (1, 3, 5 and 10% was prepared in mixture with beef, chicken and mutton. The primary CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b b (cytochrome b gene and PCR successfully amplified fragments of 359 bp. To distinguish pig species existence, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed that pig mitochondrial DNA was cut into 131 and 228 bp fragments. A polymerase chain reaction (PCR method based on the nucleotide sequence variation in the amelogenin gene has been chosen for the specific identification of pork DNAs in mixture meat. The primers designed generated specific fragments of 353 and 312 bp length for pork. The specificity of the primary designed was tested on 4 animal species including pig, cattle, chicken and goat species. Analysis of experimental mixture meat demonstrated that 1% of raw pork tissues could be detected using PCR-RFLP with BseDI restriction enzyme but detection using species-specific PCR showed the cross reactivity to beef, chicken and mutton. The cytochrome b PCR-RFLP species identification assay yielded excellent results for identification of pig species. PCR-RFLP is a potentially reliable technique for detection of the existence of pork in animal food product for Halal authentication. Keywords: Pork identification, cytochrome b, amelogenin, polymerase chain reaction   ABSTRAK   Penelitian ini dilakukan untuk mengaplikasikan metode deteksi daging babi dalam campuan daging dengan sapi, kambing dan ayam melalui PCR-RFLP dan PCR dengan primer spesifik untuk babi. Level kontaminasi daging babi dibuat sebesar 1, 3, 5 dan 10% dari total daging dalam campuran. Metode PCR-RFLP menggunakan sepasang primer yaitu gen cytochrome b dari mitokondria yang

Role of Asp544 in subunit I for Na+ pumping by Vitreoscilla cytochrome bo

International Nuclear Information System (INIS)

Chung, Yeon T.; Stark, Benjamin C.; Webster, Dale A.

2006-01-01

The conserved Glu540 in subunit I of Escherichia coli cytochrome bo (a H + pump) is replaced by Asp544 in the Vitreoscilla enzyme (a Na + pump). Site-directed mutagenesis of the Vitreoscilla cytochrome bo operon changed this Asp to Glu, and both wild type and mutant cyo's were transformed into E. coli strain GV100, which lacks cytochrome bo. Compared to the wild type transformant the Asp544Glu transformant had decreased ability to pump Na + as well as decreased stimulation in respiratory activity in the presence of Na + . Preliminary experiments indicated that this mutant also had increased ability to pump protons, suggesting that this single change may provide cation pumping specificity in this group of enzymes

Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data

International Nuclear Information System (INIS)

Dosset, Patrice; Hus, Jean-Christophe; Blackledge, Martin; Marion, Dominique

2000-01-01

A novel program has been developed for the interpretation of 15 N relaxation rates in terms of macromolecular anisotropic rotational diffusion. The program is based on a highly efficient simulated annealing/minimization algorithm, designed specifically to search the parametric space described by the isotropic, axially symmetric and fully anisotropic rotational diffusion tensor models. The high efficiency of this algorithm allows extensive noise-based Monte Carlo error analysis. Relevant statistical tests are systematically applied to provide confidence limits for the proposed tensorial models. The program is illustrated here using the example of the cytochrome c' from Rhodobacter capsulatus, a four-helix bundle heme protein, for which data at three different field strengths were independently analysed and compared

Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis

International Nuclear Information System (INIS)

Hiraoka, Yoshinori; Granja, Ana Teresa; Fialho, Arsenio M.; Schlarb-Ridley, Beatrix G.; Das Gupta, Tapas K.; Chakrabarty, Ananda M.; Yamada, Tohru

2005-01-01

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c 551 from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c 551 , the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G 1 to S phase, as well as at the G 2 /M phase at higher concentrations. Unlike P. aeruginosa cytochrome c 551 which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G 2 /M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 deg. C, suggesting energy requirement in the entry process

Cytochrome oxidase as an indicator of ice storage and frozen storage

DEFF Research Database (Denmark)

Godiksen, Helene; Jessen, Flemming

2001-01-01

in different cods was 21%, and the coefficient of variation of different analyses on the same homogenate was 5%. It was shown that ice storage of muscle samples before they were frozen and thawed resulted in a major freezing-induced activation of cytochrome oxidase activity. The enzyme may therefore be used...... as an indicator of frozen fish to determine if the fish has been stored on ice before freezing. Cytochrome oxidase activity showed also potential as an indicator of frozen storage, as it was possible to distinguish between the frozen storage temperatures -9, -20, and -40 degreesC....

Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.

Science.gov (United States)

González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A

2015-08-11

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ's histone chaperone activity.

Rieske iron-sulfur protein of the cytochrome bc(1) complex: a potential target for fungicide discovery.

Science.gov (United States)

Yang, Wen-Chao; Li, Hui; Wang, Fu; Zhu, Xiao-Lei; Yang, Guang-Fu

2012-07-23

The cytochrome bc(1) complex (complex III, cyt bc(1)) is an essential component of cellular respiration. Cyt bc(1) has three core subunits that are required for its catalytic activity: cytochrome b, cytochrome c(1), and the Rieske iron-sulfur protein (ISP). Although most fungicides inhibit this enzyme by binding to the cytochrome b subunit, resistance to these fungicides has developed rapidly due to their widespread application. Resistance is mainly associated with mutations in cytochrome b, the only subunit encoded by mitochondrial DNA. Recently, the flexibility and motion of the ISP and its essential role in electron transfer have received intense attention; this leads us to propose a new classification of cyt bc(1) inhibitors (three types of Q(o) inhibitors) that mobilize, restrict, or fix the rotation of the ISP. Importantly, the strengths of the ISP-inhibitor interactions correlate with inhibitor activity and the development of resistance to Q(o) inhibitors, thereby offering clues for designing novel cyt bc(1) inhibitors with high potency and a low risk of resistance. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Covalent modification of cytochrome c by reactive metabolites of furan.

Science.gov (United States)

Phillips, Martin B; Sullivan, Mathilde M; Villalta, Peter W; Peterson, Lisa A

2014-01-21

Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivity of these two reactive intermediates, cytochrome c was reacted with BDA in the presence and absence of GSH. As judged by MALDI-TOF mass spectrometry, BDA reacts extensively with cytochrome c to form adducts that add 66 Da to the protein, consistent with the formation of pyrrolinone adducts. Addition of GSH to the reaction mixture reduced the overall extent of adduct formation. The mass of the adducted protein was shifted by 355 Da as expected for GSH-BDA-protein cross-link formation. LC-MS/MS analysis of the tryptic digests of the alkylated protein indicated that the majority of adducts occurred on lysine residues, with BDA reacting less selectively than GSH-BDA. Both types of adducts may contribute to the toxic effects of furan.

Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation.

Science.gov (United States)

Yun, Jiae; Malvankar, Nikhil S; Ueki, Toshiyuki; Lovley, Derek R

2016-02-01

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.

Dimers of light-harvesting complex 2 from Rhodobacter sphaeroides characterized in reconstituted 2D crystals with atomic force microscopy

NARCIS (Netherlands)

Liu, Lu-Ning; Aartsma, Thijs J.; Frese, Raoul N.

Microscopic and light spectroscopic investigations on the supramolecular architecture of bacterial photosynthetic membranes have revealed the photosynthetic protein complexes to be arranged in a densely packed energy-transducing network. Protein packing may play a determining role in the formation

Kinetics and Mechanistic Studies on the Reaction between Cytochrome c and Tea Catechins

Directory of Open Access Journals (Sweden)

Lihua Wang

2014-08-01

Full Text Available Green tea is characterized by the presence of an abundance of polyphenolic compounds, also known as catechins, including epicatechin (EC, epigallocatechin (EGC, epicatechin gallate (EGC and epigallocatechin gallate (EGCG. In addition to being a popular beverage, tea consumption has been suggested as a mean of chemoprevention. However, its mode of action is unclear. It was discovered that tea catechins can react with cytochrome c. When oxidized cytochrome c was mixed with catechins commonly found in green tea under non-steady-state conditions, a reduction of cytochrome c was observed. The reaction rate of the catechins was dependent on the pH and the nature of the catechin. The pseudo-first order rate constant obtained increased in the order of EC < ECG < EGC < EGCG, which is consistent with previously reported superoxide reduction activities and Cu2+ reduction activities of tea catechins.

Potential of Rhodobacter capsulatus Grown in Anaerobic-Light or Aerobic-Dark Conditions as Bioremediation Agent for Biological Wastewater Treatments

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Stefania Costa

2017-02-01

Full Text Available The use of microorganisms to clean up wastewater provides a cheaper alternative to the conventional treatment plant. The efficiency of this method can be improved by the choice of microorganism with the potential of removing contaminants. One such group is photosynthetic bacteria. Rhodobacter capsulatus is a purple non-sulfur bacterium (PNSB found to be capable of different metabolic activities depending on the environmental conditions. Cell growth in different media and conditions was tested, obtaining a concentration of about 108 CFU/mL under aerobic-dark and 109 CFU/mL under anaerobic-light conditions. The biomass was then used as a bioremediation agent for denitrification and nitrification of municipal wastewater to evaluate the potential to be employed as an additive in biological wastewater treatment. Inoculating a sample of mixed liquor withdrawn from the municipal wastewater treatment plant with R. capsulatus grown in aerobic-dark and anaerobic-light conditions caused a significant decrease of N-NO3 (>95%, N-NH3 (70% and SCOD (soluble chemical oxygen demand (>69%, independent of the growth conditions. A preliminary evaluation of costs indicated that R. capsulatus grown in aerobic-dark conditions could be more convenient for industrial application.

Identification of human cytochrome P450s as autoantigens.

Science.gov (United States)

Manns, M P; Johnson, E F

1991-01-01

Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

Molecular Computational Investigation of Electron Transfer Kinetics across Cytochrome-Iron Oxide Interfaces

International Nuclear Information System (INIS)

Kerisit, Sebastien N.; Rosso, Kevin M.; Dupuis, Michel; Valiev, Marat

2007-01-01

The interface between electron transfer proteins such as cytochromes and solid phase mineral oxides is central to the activity of dissimilatory-metal reducing bacteria. A combination of potential-based molecular dynamics simulations and ab initio electronic structure calculations are used in the framework of Marcus' electron transfer theory to compute elementary electron transfer rates from a well-defined cytochrome model, namely the small tetraheme cytochrome (STC) from Shewanella oneidensis, to surfaces of the iron oxide mineral hematite (a-Fe2O3). Room temperature molecular dynamics simulations show that an isolated STC molecule favors surface attachment via direct contact of hemes I and IV at the poles of the elongated axis, with electron transfer distances as small as 9 Angstroms. The cytochrome remains attached to the mineral surface in the presence of water and shows limited surface diffusion at the interface. Ab initio electronic coupling matrix element (VAB) calculations of configurations excised from the molecular dynamics simulations reveal VAB values ranging from 1 to 20 cm-1, consistent with nonadiabaticity. Using these results, together with experimental data on the redox potential of hematite and hemes in relevant cytochromes and calculations of the reorganization energy from cluster models, we estimate the rate of electron transfer across this model interface to range from 1 to 1000 s-1 for the most exothermic driving force considered in this work, and from 0.01 to 20 s-1 for the most endothermic. This fairly large range of electron transfer rates highlights the sensitivity of the rate upon the electronic coupling matrix element, which is in turn dependent on the fluctuations of the heme configuration at the interface. We characterize this dependence using an idealized bis-imidazole heme to compute from first principles the VAB variation due to porphyrin ring orientation, electron transfer distance, and mineral surface termination. The electronic

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase

NARCIS (Netherlands)

van Kuilenburg, A. B.; Gorren, A. C.; Dekker, H. L.; Nieboer, P.; van Gelder, B. F.; Muijsers, A. O.

1992-01-01

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of

On the role of cytochrome c8 in photosynthetic electron transfer of the purple non-sulfur bacterium Rhodoferax fermentans

DEFF Research Database (Denmark)

Hochkoeppler, Alejandro; Ciurli, Stefano; Kofod, Pauli

1997-01-01

We report on the isolation, purification and functional characterization of a soluble c-type cytochrome from light-grown cells of the purple phototroph Rhodoferax fermentans. This cytochrome is basic (pI = 8), has a molecular mass of 12 kDa, and is characterized by a midpoint reduction potential...... center, in a fast (sub-ms) and a slow (ms) phase. Competition experiments in the presence of both cytochrome c8 and high potential iron-sulfur protein (HiPIP), isolated from the same microorganism, show that cytochrome c8 oxidation is decreased upon addition of HiPIP. These observations suggest...

Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing

International Nuclear Information System (INIS)

Serra, A.S.; Jorge, S.R.; Silveira, C.M.; Moura, J.J.G.; Jubete, E.; Ochoteco, E.; Cabanero, G.; Grande, H.; Almeida, M.G.

2011-01-01

In this work, a novel enzymatic biosensor for determination of nitrites constructed on an electrochemical transducing platform is proposed. The sensor is based on cytochrome-cd 1 (cyt-cd 1 ) nitrite reductase from Marinobacter hydrocarbonoclasticus strain 617 as biological recognition element, and its putative physiological redox partner cytochrome-c 552 (cyt-c 552 ), as electron mediator. The proteins were co-immobilized using a photopolymerizable polyvinyl alcohol (PVA) derivative, onto carbon paste screen printed electrodes (CPSPEs); the optimal modification conditions were 100 μM cyt-cd 1 /100 μM cyt-c 552 and 50% PVA, after a 48 h polymerization time. Electrochemical characterization of the mediator was carried out by cyclic voltammetry. The one-electron exchange between cyt-c 552 and the working electrode is a quasi-reversible process, without mass transport limitations. The formal potential of the mediator is 254 ± 2 mV vs NHE and the intermolecular electron transfer rate constant between cytochromes c 552 and cd 1 is 9.9 x 10 3 M -1 s -1 . The analytical parameters of the biosensor response to nitrite as assessed by amperometric measurements were: linear range from 10 to 200 μM; detection and quantification limits of 7 and 24 μM, respectively; sensitivity of 2.49 ± 0.08 A mol -1 cm 2 μM -1 . Catalytic profiles in the presence of possible interfering species were also investigated. The interference from competitive enzymatic reduction of dissolved oxygen could be overcome by tuning the cyclic voltammograms for faster sweep rates.

Cytochrome c biosensor for determination of trace levels of cyanide and arsenic compounds

International Nuclear Information System (INIS)

Fuku, Xolile; Iftikar, Faiza; Hess, Euodia; Iwuoha, Emmanuel; Baker, Priscilla

2012-01-01

Highlights: ► Cytochrome c biosensor for detection of KCN, As 2 O 3 and Fe 2 K (CN) was constructed. ► Detection limits in the range of 4.3–9.1 μM for the analytes were obtained using CV, SWV and EIS. ► The detection limits for the biosensor were significantly lower than current EPA and WHO guidelines. - Abstract: An electrochemical method based on a cytochrome c biosensor was developed, for the detection of selected arsenic and cyanide compounds. Boron doped diamond (BDD) electrode was used as a transducer, onto which cytochrome c was immobilised and used for direct determination of Prussian blue, potassium cyanide and arsenic trioxide. The sensitivity as calculated from cyclic voltammetry (CV) and square wave voltammetry (SWV), for each analyte in phosphate buffer (pH = 7) was found to be in the range of (1.1–4.5) × 10 −8 A μM −1 and the detection limits ranged from 4.3 to 9.1 μM. The biosensor is therefore able to measure significantly lower than current Environmental Protection Agency (EPA) and World Health Organisation (WHO) guidelines, for these types of analytes. The protein binding was monitored as a decrease in biosensor peak currents by SWV and as an increase in biosensor charge transfer resistance by electrochemical impedance spectroscopy (EIS). EIS provided evidence that the electrocatalytic advantage of BDD electrode was not lost upon immobilisation of cytochrome c. The interfacial kinetics of the biosensor was modelled as equivalent electrical circuit based on electrochemical impedance spectroscopy data. UV–vis spectroscopy was used to confirm the binding of the protein in solution by monitoring the intensity of the soret bands and the Q bands. FTIR was used to characterise the protein in the immobilised state and to confirm that the protein was not denatured upon binding to the pre-treated bare BDD electrode. SNFTIR of cyt c immobilised at platinum electrode, was used to study the effect of oxidation state on the surface bond

Removal of the effect of ammonium on the regulation of nitrogenase enzyme in Rhodobacter capsulatus DSM1710 for improved hydrogen production

Energy Technology Data Exchange (ETDEWEB)

Pekgoez, Guelsah; Guenduez, Ufuk [Middle East Technical Univ. (Turkey). Dept. of Biology; Eroglu, Inci [Middle East Technical Univ. (Turkey). Dept. of Chemical Engineering; Rakhely, Gabor [Szeged Univ. (Hungary). Dept. of Biotechnology

2010-07-01

Photofermentative biohydrogen production by purple non-sulfur (PNS) bacteria is a renewable and clean way of producing hydrogen. Hydrogen production by PNS bacteria, Rhodobacter capsulatus, is mediated mainly by nitrogenases, which primarily fix molecular nitrogen to ammonium and produce hydrogen as byproduct. The reaction catalyzed by nitrogenases requires a lot of energy. Hence, there is a complex regulation on nitrogenase enzyme complex, consequently, on hydrogen production. Whenever ammonium, which is the end product of nitrogen fixation reaction, is found in the environment, hydrogen production stops. GlnB and GlnK proteins are the critical regulatory proteins in ammonium dependent regulation of the nitrogenase gene expression. In this study, the aim is to release the ammonium regulation on nitrogenase enzyme by inactivating glnB and glnK genes. For this purpose, relevant recombinant vectors were constructed; R.capsulatus glnB- strain was obtained. The double R.capsulatus glnB{sup -}glnK{sup -} strain, able to produce hydrogen independent of ammonium concentration of the environment is to be obtained. (orig.)

Biomimetic Membranes for Multi-Redox Center Proteins

Directory of Open Access Journals (Sweden)

Renate L. C. Naumann

2016-03-01

Full Text Available His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs. An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM. MRPs, such as the cytochrome c oxidase (CcO from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS and surface-enhanced resonance Raman spectroscopy (SERRS, were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs. The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

Cytochrome P-450 dependent ethanol oxidation. Kinetic isotope effects and absence of stereoselectivity

International Nuclear Information System (INIS)

Ekstroem, G.; Norsten, C.; Cronholm, T.; Ingelman-Sundberg, M.

1987-01-01

Deuterium isotope effects [/sup D/(V/K)] and stereoselectivity of ethanol oxidation in cytochrome P-450 containing systems and in the xanthine-xanthine oxidase system were compared with those of yeast alcohol dehydrogenase. The isotope effects were determined by using both a noncompetitive method, including incubation of unlabeled of [1,1- 2 H 2 ] ethanol at various concentrations, and a competitive method, where 1:1 mixtures of [1- 13 C]- and [ 2 H 6 ] ethanol or [2,2,2- 2 H 3 ]- and [1,1- 2 H 2 ] ethanol were incubated and the acetaldehyde formed was analyzed by gas chromatography/mass spectrometry. The /sup D/(V/K) isotope effects of the cytochrome P-450 dependent ethanol oxidation were about 4 with liver microsomes from imidazole-, phenobarbital- or acetone-treated rabbits or with microsomes from acetone- or ethanol-treated rats. Similar isotope effects were reached with reconstituted membranes containing the rabbit ethanol-inducible cytochrome P-450 (LMeb), whereas control rat microsomes and membranes containing rabbit phenobarbital-inducible P-450 LM 2 oxidized the alcohol with /sup D/(V/K) of about 2.8 and 1.8, respectively. Addition of Fe/sup III/EDTA either to microsomes from phenobarbital-treated rabbits or to membranes containing P-450 LMeb significantly lowered the isotope effect. Incubations of all cytochrome P-450 containing systems of the xanthine-xanthine oxidase systems with (1R)- and (1S)-[1- 2 H] ethanol, revealed, taking the isotope effects into account, that 44-66% of the ethanol oxidized had lost the 1-pro-R hydrogen. The data indicate that cytochrome P-450 dependent ethanol oxidation is not stereospecific and that cleavage of the C 1 -H bond appears to be a rate-determining step in the catalysis by the ethanol-inducible form of P-450. The contribution of hydroxyl radicals in ethanol oxidation by the various enzymic systems is discussed

In situ Raman study of redox state changes of mitochondrial cytochromes in a perfused rat heart

DEFF Research Database (Denmark)

Brazhe, Nadezda; Treiman, Marek; Faricelli, Barbara

2013-01-01

We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial...... surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling...

Reduction of chalcogen oxyanions and generation of nanoprecipitates by the photosynthetic bacterium Rhodobacter capsulatus

Energy Technology Data Exchange (ETDEWEB)

Borghese, Roberto, E-mail: roberto.borghese@unibo.it [Department of Pharmacy and Biotechnology, University of Bologna (Italy); Baccolini, Chiara; Francia, Francesco [Department of Pharmacy and Biotechnology, University of Bologna (Italy); Sabatino, Piera [Department of Chemistry G. Ciamician, University of Bologna (Italy); Turner, Raymond J. [Department of Biological Sciences, University of Calgary, Calgary, Alberta (Canada); Zannoni, Davide, E-mail: davide.zannoni@unibo.it [Department of Pharmacy and Biotechnology, University of Bologna (Italy)

2014-03-01

Graphical abstract: - Highlights: • R. capsulatus cells produce extracellular chalcogens nanoprecipitates when lawsone is present. • Lawsone acts as a redox mediator from reducing equivalents to tellurite and selenite. • Nanoprecipitates production depends on carbon source and requires metabolically active cells. • Te{sup 0} and Se{sup 0} nanoprecipitates are identified by X-ray diffraction (XRD) spectroscopy. - Abstract: The facultative photosynthetic bacterium Rhodobacter capsulatus is characterized in its interaction with the toxic oxyanions tellurite (Te{sup IV}) and selenite (Se{sup IV}) by a highly variable level of resistance that is dependent on the growth mode making this bacterium an ideal organism for the study of the microbial interaction with chalcogens. As we have reported in the past, while the oxyanion tellurite is taken up by R. capsulatus cells via acetate permease and it is reduced to Te{sup 0} in the cytoplasm in the form of splinter-like black intracellular deposits no clear mechanism was described for Se{sup 0} precipitation. Here, we present the first report on the biotransformation of tellurium and selenium oxyanions into extracellular Te{sup 0} and Se{sup 0}nanoprecipitates (NPs) by anaerobic photosynthetically growing cultures of R. capsulatus as a function of exogenously added redox-mediator lawsone, i.e. 2-hydroxy-1,4-naphthoquinone. The NPs formation was dependent on the carbon source used for the bacterial growth and the rate of chalcogen reduction was constant at different lawsone concentrations, in line with a catalytic role for the redox mediator. X-ray diffraction (XRD) analysis demonstrated the Te{sup 0} and Se{sup 0} nature of the nanoparticles.

Role of cytochrome B in the processing of the subunits of complex III in the yeast mitochondria

International Nuclear Information System (INIS)

Sen, K.G.

1986-01-01

The work described in this dissertation deals with the effect of cytochrome b on the biogenesis and assembly of the subunits of complex III in the mitochondrial membrane of the yeast Saccharomyces cerevisiae. The cytochrome b-mutants (Box mutants of S. cerevisiae form an excellent system to study such a role of cytochome B. The amounts of cytochrome c 1 in the mitochrondria, as determined both spectroscopically and immunologically, were not affected by the absence of cytochrome b. Pulse labelling of the cells with ( 35 S) methionine in the presence of CCCP showed the accumulation of the precursors to the core protein I and the iron-sulfur protein in similar amounts in the mutant Box 6-2 and the wild type cells. Synthesis of the iron sulfur protein and the cytochrome c 1 by in vitro translation of mRNA isolated from wild type and mutant Box 6-2 in a rabbit reticulocyte lysate system, also confirmed that the synthesis of the nuclear encoded subunits was not affected in the mutants. Pulse labeling of the cells in the absence of CCCP and subsequent chase with cold methionine, however, showed much less of the mature subunits of core protein I and the iron-sulfur protein in the mitochrondria of the mutant cells relative to the wild type. These results indicate that cytochrome b is necessary for the proper processing of certain subunits of complex III

Lansoprazole is an antituberculous prodrug targeting cytochrome bc1.

Science.gov (United States)

Rybniker, Jan; Vocat, Anthony; Sala, Claudia; Busso, Philippe; Pojer, Florence; Benjak, Andrej; Cole, Stewart T

2015-07-09

Better antibiotics capable of killing multi-drug-resistant Mycobacterium tuberculosis are urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity against M. tuberculosis. Ex vivo pharmacokinetics and target identification studies reveal that lansoprazole kills M. tuberculosis by targeting its cytochrome bc1 complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochrome bc1 inhibitors is highly active against drug-resistant clinical isolates and spares the human H(+)K(+)-ATPase thus providing excellent opportunities for targeting the major pathogen M. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens.

Variations in epidermal cytochrome oxidase activity after local irradiation

International Nuclear Information System (INIS)

Itoiz, M.E.; Rey, B.M. de; Cabrini, R.L.

1982-01-01

Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16krad. The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied. At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles. (author)

Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats

International Nuclear Information System (INIS)

Das, M.; Dixit, R.; Mukhtar, H.; Bickers, D.R.

1985-01-01

The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H 2 O 2 such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization

Evidence from the structure and function of cytochromes c(2) that nonsulfur purple bacterial photosynthesis followed the evolution of oxygen respiration.

Science.gov (United States)

Meyer, Terry; Van Driessche, Gonzalez; Ambler, Richard; Kyndt, John; Devreese, Bart; Van Beeumen, Jozef; Cusanovich, Michael

2010-10-01

Cytochromes c(2) are the nearest bacterial homologs of mitochondrial cytochrome c. The sequences of the known cytochromes c(2) can be placed in two subfamilies based upon insertions and deletions, one subfamily is most like mitochondrial cytochrome c (the small C2s, without significant insertions and deletions), and the other, designated large C2, shares 3- and 8-residue insertions as well as a single-residue deletion. C2s generally function between cytochrome bc(1) and cytochrome oxidase in respiration (ca 80 examples known to date) and between cytochrome bc(1) and the reaction center in nonsulfur purple bacterial photosynthesis (ca 21 examples). However, members of the large C2 subfamily are almost always involved in photosynthesis (12 of 14 examples). In addition, the gene for the large C2 (cycA) is associated with those for the photosynthetic reaction center (pufBALM). We hypothesize that the insertions in the large C2s, which were already functioning in photosynthesis, allowed them to replace the membrane-bound tetraheme cytochrome, PufC, that otherwise mediates between the small C2 or other redox proteins and photosynthetic reaction centers. Based upon our analysis, we propose that the involvement of C2 in nonsulfur purple bacterial photosynthesis was a metabolic feature subsequent to the evolution of oxygen respiration.

Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study

International Nuclear Information System (INIS)

Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Tavagnacco, Claudio; Borsari, Marco

2011-01-01

Highlights: → Denaturation involves intermediate and partially unfolded forms. → An unfolded species displaying the haem with Fe coordinated by two His is observed. → Under unfolding conditions the nature of the SAM influences conformation of protein. → Concentration of the unfolding agent affects redox properties of immobilized protein. - Abstract: The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E o ' of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E o ' value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions.

Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study

Energy Technology Data Exchange (ETDEWEB)

Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia [Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena (Italy); Tavagnacco, Claudio [Department of Chemistry, University of Trieste, via Giorgieri 1, 34127 Trieste (Italy); Borsari, Marco, E-mail: marco.borsari@unimore.it [Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena (Italy)

2011-08-01

Highlights: > Denaturation involves intermediate and partially unfolded forms. > An unfolded species displaying the haem with Fe coordinated by two His is observed. > Under unfolding conditions the nature of the SAM influences conformation of protein. > Concentration of the unfolding agent affects redox properties of immobilized protein. - Abstract: The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E{sup o}' of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E{sup o}' value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions.

Zolpidem metabolism in vitro: responsible cytochromes, chemical inhibitors, and in vivo correlations

Science.gov (United States)

von Moltke, Lisa L; Greenblatt, David J; Granda, Brian W; Duan, Su Xiang; Grassi, Jeffrey M; Venkatakrishnan, Karthik; Harmatz, Jerold S; Shader, Richard I

1999-01-01

Aims To determine the human cytochromes mediating biotransformation of the imidazopyridine hypnotic, zolpidem, and the clinical correlates of the findings. Methods Kinetic properties of zolpidem biotransformation to its three hydroxylated metabolites were studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. Results The metabolic product termed M-3 accounted for more than 80% of net intrinsic clearance by liver microsomes in vitro. Microsomes containing human cytochromes CYP1A2, 2C9, 2C19, 2D6, and 3 A4 expressed by cDNA-transfected human lymphoblastoid cells mediated zolpidem metabolism in vitro. The kinetic profile for zolpidem metabolite formation by each individual cytochrome was combined with estimated relative abundances based on immunological quantification, yielding projected contributions to net intrinsic clearance of: 61% for 3 A4, 22% for 2C9, 14% for 1A2, and less than 3% for 2D6 and 2C19. These values were consistent with inhibitory effects of ketoconazole and sulfaphenazole on zolpidem biotransformation by liver microsomes. Ketoconazole had a 50% inhibitory concentration (IC50) of 0.61 μm vs formation of the M-3 metabolite of zolpidem in vitro; in a clinical study, ketoconazole coadministration reduced zolpidem oral clearance by ≈40%, somewhat less than anticipated based on the IC50 value and total plasma ketoconazole levels, but much more than predicted based on unbound plasma ketoconazole levels. Conclusions The incomplete dependence of zolpidem clearance on CYP3A activity has clinical implications for susceptibility to metabolic inhibition. PMID:10383565

Solvation effect of bacteriochlorophyll excitons in light-harvesting complex LH2.

Science.gov (United States)

Urboniene, V; Vrublevskaja, O; Trinkunas, G; Gall, A; Robert, B; Valkunas, L

2007-09-15

We have characterized the influence of the protein environment on the spectral properties of the bacteriochlorophyll (Bchl) molecules of the peripheral light-harvesting (or LH2) complex from Rhodobacter sphaeroides. The spectral density functions of the pigments responsible for the 800 and 850 nm electronic transitions were determined from the temperature dependence of the Bchl absorption spectra in different environments (detergent micelles and native membranes). The spectral density function is virtually independent of the hydrophobic support that the protein experiences. The reorganization energy for the B850 Bchls is 220 cm(-1), which is almost twice that of the B800 Bchls, and its Huang-Rhys factor reaches 8.4. Around the transition point temperature, and at higher temperatures, both the static spectral inhomogeneity and the resonance interactions become temperature-dependent. The inhomogeneous distribution function of the transitions exhibits less temperature dependence when LH2 is embedded in membranes, suggesting that the lipid phase protects the protein. However, the temperature dependence of the fluorescence spectra of LH2 cannot be fitted using the same parameters determined from the analysis of the absorption spectra. Correct fitting requires the lowest exciton states to be additionally shifted to the red, suggesting the reorganization of the exciton spectrum.

Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

Science.gov (United States)

Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

2016-11-01

Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes.

Science.gov (United States)

Swainsbury, David J K; Scheidelaar, Stefan; Foster, Nicholas; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2017-10-01

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

The Iron-Sulfur Cluster of Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO) is the Electron Acceptor for Electron Transfer Flavoprotein†

Science.gov (United States)

Swanson, Michael A.; Usselman, Robert J.; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

2011-01-01

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone-pool. It contains one [4Fe-4S]2+,1+ and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S]2+,1+ to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S]+ at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 mV and −30 mV for wild type to −11 mV and −19 mV, respectively. The N338A mutation decreased the potentials to −37 mV and −49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e− catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone, but not in electron transfer from ETF to ETF-QO. Therefore the iron-sulfur cluster is the immediate acceptor from ETF. PMID:18672901

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

Energy Technology Data Exchange (ETDEWEB)

Jan, Yi-Hua [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Baker, Angela A. [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Mishin, Vladimir [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Heck, Diane E. [Department of Environmental Health Science, New York Medical College, Valhalla, NY (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States)

2015-10-01

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

International Nuclear Information System (INIS)

Jan, Yi-Hua; Richardson, Jason R.; Baker, Angela A.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

2015-01-01

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.

Supercapacitors based on c-type cytochromes using conductive nanostructured networks of living bacteria.

Science.gov (United States)

Malvankar, Nikhil S; Mester, Tünde; Tuominen, Mark T; Lovley, Derek R

2012-02-01

Supercapacitors have attracted interest in energy storage because they have the potential to complement or replace batteries. Here, we report that c-type cytochromes, naturally immersed in a living, electrically conductive microbial biofilm, greatly enhance the device capacitance by over two orders of magnitude. We employ genetic engineering, protein unfolding and Nernstian modeling for in vivo demonstration of charge storage capacity of c-type cytochromes and perform electrochemical impedance spectroscopy, cyclic voltammetry and charge-discharge cycling to confirm the pseudocapacitive, redox nature of biofilm capacitance. The biofilms also show low self-discharge and good charge/discharge reversibility. The superior electrochemical performance of the biofilm is related to its high abundance of cytochromes, providing large electron storage capacity, its nanostructured network with metallic-like conductivity, and its porous architecture with hydrous nature, offering prospects for future low cost and environmentally sustainable energy storage devices. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

Czech Academy of Sciences Publication Activity Database

Sanderson, T. H.; Mahapatra, G.; Pecina, Petr; Ji, Q.; Yu, K.; Sinkler, Ch.; Varughese, A.; Kumar, R.; Bukowski, M. J.; Tousignant, R. N.; Salomon, A. R.; Lee, I.; Hüttemann, M.

2013-01-01

Roč. 8, č. 11 (2013), e78627 E-ISSN 1932-6203 Institutional support: RVO:67985823 Keywords : cytochrome c * tyrosine phosphorylation * brain ischemia * insulin Subject RIV: ED - Physiology Impact factor: 3.534, year: 2013

Computational identification of putative cytochrome P450 genes in ...

African Journals Online (AJOL)

Chattha

Economically, legumes represent the second most important family of crop plants after Poacea (grass family), accounting for ... further characterization of P450 genes with both known and unknown functions. MATERIALS AND METHODS ..... Cytochrome P450. In: Somerville CR, Meyerowitz EM (eds) .The Arabidopsis book,.

Multivariate Modeling of Cytochrome P450 Enzymes for 4 ...

African Journals Online (AJOL)

Conclusion: Apart from insights into important molecular properties for CYP inhibition, the findings may also guide further investigations of novel drug candidates that are unlikely to inhibit multiple CYP sub-types. Keywords: Antimalarial, Chloroquine, Cytochrome P450, Genetic algorithm-based multiple linear regression, ...

Molecular Characterization and Functional Analysis of Three Pathogenesis-Related Cytochrome P450 Genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea

Directory of Open Access Journals (Sweden)

Xiao-Lu Xu

2015-03-01

Full Text Available Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c

International Nuclear Information System (INIS)

Ilan, Y.; Shafferman, A.; Feinberg, B.A.; Lau, Y.K.

1979-01-01

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH 3 CHOH, CO 2 , O 2 , and e - /sub aq/, and the oxidation of the reduced cytochrome c derivatives by Fe(CN) 6 3- were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partitioned from each other in some cases. In regard to cycochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region

Relation among cytochrome P450, AH-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

Science.gov (United States)

Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

1994-01-01

Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P-450-associated monooxygenases and cytochrome P-450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r-2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

The effect of amixin and agmatine on cytochrome c release from isolated mitochondria

Directory of Open Access Journals (Sweden)

K. R. Uspenska

2017-02-01

Full Text Available Mitochondrial nicotinic acetylcholine receptors (nAChRs control permeability transition pore formation and cytochrome c release in the presence of apoptogenic factors. This study demonstrates that pharmacological agents amixin and agmatine affect mitochondrial nAChR functioning: they slightly suppress cytochrome c release from mouse brain and liver mitochondria stimulated with apoptogenic dose of Са2+ and prevent the effect of α7 nAChR agonist PNU282987. We conclude that mitochondria may be one of therapeutic targets of amixin and agmatine.

Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing

DEFF Research Database (Denmark)

Andersen, Trine Bundgaard; Hansen, Niels Bjørn; Laursen, Tomas

2016-01-01

The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to ...... (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes....

Construction and engineering of a thermostable self-sufficient cytochrome P450

Energy Technology Data Exchange (ETDEWEB)

Mandai, Takao; Fujiwara, Shinsuke [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan); Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)

2009-06-19

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

Construction and engineering of a thermostable self-sufficient cytochrome P450

International Nuclear Information System (INIS)

Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

2009-01-01

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP + reductase (FNR): H 2 N-CYP175A1-Fdx-FNR-COOH (175FR) and H 2 N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 o C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

Hydrogen/deuterium exchange of multiply-protonated cytochrome c ions

International Nuclear Information System (INIS)

Wood, T.D.; Guan, Ziqiang; O'Connor, P.B.

1995-01-01

Low resolution measurements show gaseous multiply-protonated cytochrome c ions undergo hydrogen/deuterium (H/D) exchange with pseudo first-order kinetics at three distinct exchange levels, suggesting the co-existence of gaseous protein conformations. Although exchange levels first increase with increasing charge values, they decrease at the highest charge values, consistent with solution-phase behavior of cytochrome c, where the native structure unfolds with decreasing pH until folding into a compact A-state at lowest pH. High resolution measurements indicate the presence of at least six H/D exchange levels. Infrared (IR) laser heating and fast collisions via quadrupolar excitation (QE) increase H/D exchange levels (unfolding) while charge-stripping ions to lower charge values can increase or decrease H/D exchange levels (unfolding or folding). Wolynes has suggested studying proteins in vacuo could play an important role in delineating the contributions various forces play in the protein folding process, provided appropriate comparisons can be made between gas-phase and solution-phase structures

Natively oxidized amino acid residues in the spinach cytochrome b 6 f complex.

Science.gov (United States)

Taylor, Ryan M; Sallans, Larry; Frankel, Laurie K; Bricker, Terry M

2018-01-29

The cytochrome b 6 f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b 6 f is 10-20 fold higher than that observed for the analogous respiratory cytochrome bc 1 complex. The types of ROS produced (O 2 •-, 1 O 2 , and, possibly, H 2 O 2 ) and the site(s) of ROS production within the b 6 f complex have been the subject of some debate. Proposed sources of ROS have included the heme b p , PQ p •- (possible sources for O 2 •- ), the Rieske iron-sulfur cluster (possible source of O 2 •- and/or 1 O 2 ), Chl a (possible source of 1 O 2 ), and heme c n (possible source of O 2 •- and/or H 2 O 2 ). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b 6 f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b 6 f complex.

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

NARCIS (Netherlands)

Schallmey, Anett; den Besten, Gijs; Teune, Ite G. P.; Kembaren, Roga F.; Janssen, Dick B.

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The

An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications.

Science.gov (United States)

Brixius-Anderko, Simone; Hannemann, Frank; Ringle, Michael; Khatri, Yogan; Bernhardt, Rita

2017-05-01

Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 μM, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Versatility of non-native forms of human cytochrome c: pH and micellar concentration dependence.

Science.gov (United States)

Simon, Matthieu; Metzinger-Le Meuth, Valérie; Chevance, Soizic; Delalande, Olivier; Bondon, Arnaud

2013-01-01

In addition to its electron transfer activity, cytochrome c is now known to trigger apoptosis via peroxidase activity. This new function is related to a structural modification of the cytochrome upon association with anionic lipids, particularly cardiolipin present in the mitochondrial membrane. However, the exact nature of the non-native state induced by this interaction remains an active subject of debate. In this work, using human cytochromes c (native and two single-histidine mutants and the corresponding double mutant) and micelles as a hydrophobic medium, we succeeded, through UV-visible spectroscopy, circular dichroism spectroscopy and NMR spectroscopy, in fully characterizing the nature of the sixth ligand replacing the native methionine. Furthermore, careful pH titrations permitted the identification of the amino acids involved in the iron binding over a range of pH values. Replacement of the methionine by lysine was only observed at pH above 8.5, whereas histidine binding is dependent on both pH and micelle concentration. The pH variation range for histidine protonation is relatively narrow and is consistent with the mitochondrial intermembrane pH changes occurring during apoptosis. These results allow us to rule out lysine as the sixth ligand at pH values close to neutrality and reinforce the role of histidines (preferentially His33 vs. His26) as the main candidate to replace methionine in the non-native cytochrome c. Finally, on the basis of these results and molecular dynamics simulations, we propose a 3D model for non-native cytochrome c in a micellar environment.

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

Energy Technology Data Exchange (ETDEWEB)

Ren, Jian-Ching; Rebrin, Igor [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States); Klichko, Vladimir; Orr, William C. [Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275 (United States); Sohal, Rajindar S., E-mail: sohal@usc.edu [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States)

2010-10-08

Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

International Nuclear Information System (INIS)

Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir; Orr, William C.; Sohal, Rajindar S.

2010-01-01

Research highlights: → Cytochrome c oxidase loses catalytic activity during the aging process. → Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. → Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H 2 O 2 generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

Rational redesign of the biodegradative enzyme cytochrome P450 cam:

International Nuclear Information System (INIS)

Ornstein, R.; Paulsen, M.; Bass, M.; Arnold, G.

1991-03-01

Cytochromes P450, a superfamily of monooxygenase enzymes present in all kingdoms of living organisms, are very versatile with respect to substrate range and catalytic functionality. Many recalcitrant halogenated hydrocarbons, on DOE sites and throughout the nation, result in serious environmental impact. Cytochromes P450 have been shown to be catalytically capable of, at least partial, dehalogenation of some such compounds. Clearly, however, their active site stereochemistry and related functional components are not well suited for this role because the rates of dehalogenation are generally rather modest. The evolution of modified active site and access channel structures may proceed very slowly if multiple genetic changes are simultaneously required for enzyme adaptation. Since each mutational event is by itself a rare event, a basic premise of our research is that designing multiple changes into an enzyme may be more timely than waiting for them to occur biologically either via natural selection or under laboratory-controlled conditions. Starting with available high-resolution x-ray crystal structures, molecular modeling and molecular dynamics simulations have been used to probe the basic structure/function principles and conformational fluctuations of the biodegradative enzyme, cytochrome P450cam (camphor hydroxylase from Pseudomonas putida) and active site mutants, to provide the fundamental understanding necessary for rational engineering of the enzyme for modified substrate specificity. In the present paper, we review our progress to data, in the area of molecular dynamics simulations and active site redesign of P450cam. 36 refs., 2 figs

The binding of cytochrome c to neuroglobin: A docking and surface plasmon resonance study

DEFF Research Database (Denmark)

Bønding, Signe Helbo; Henty, K.; Dingley, A.J.

2008-01-01

is associated with a small unfavourable enthalpy change (1.9 kcal mol-1) and a moderately large, favourable entropy change (14.8 cal mol-1 deg-1). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions....... one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved....... surface plasmon resonance studies provide a value of 45 μM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation...

Resonance Raman Optical Activity and Surface Enhanced Resonance Raman Optical Activity analysis of Cytochrome C

DEFF Research Database (Denmark)

Johannessen, Christian; Abdali, Salim; White, Peter C.

2007-01-01

High quality Resonance Raman (RR) and resonance Raman Optical Activity (ROA) spectra of cytochrome c were obtained in order to perform full assignment of spectral features of the resonance ROA spectrum. The resonance ROA spectrum of cytochrome c revealed a distinct spectral signature pattern due...... to resonance enhanced skeletal porphyrin vibrations, more pronounced than any contribution from the protein back-bone. Combining the intrinsic resonance enhancement of cytochrome c with surface plasmon enhancement by colloidal silver particles, the Surface Enhanced Resonance Raman Scattering (SERRS) and Chiral...... Enhanced Raman Spectroscopy (ChERS) spectra of the protein were successfully obtained at very low concentration (as low as 1 µM). The assignment of spectral features was based on the information obtained from the RR and resonance ROA spectra. Excellent agreement between RR and SERRS spectra is reported...

Significance of cytochrome P450 system responses and levels of bile fluorescent aromatic compounds in marine wildlife following oil spills

International Nuclear Information System (INIS)

Lee, R.F.; Anderson, J.W.

2005-01-01

The relationships among cytochrome P450 induction in marine wildlife species, levels of fluorescent aromatic compounds (FAC) in their bile, the chemical composition of the inducing compounds, the significance of the exposure pathway, and any resulting injury, as a consequence of exposure to crude oil following a spill, are reviewed. Fish collected after oil spills often show increases in cytochrome P450 system activity, cytochrome P4501A (CYP1A) and bile fluorescent aromatic compounds (FAC), that are correlated with exposure to polycyclic aromatic hydrocarbons (PAH) in the oil. There is also some evidence for increases in bile FAC and induction of cytochrome P450 in marine birds and mammals after oil spills. However, when observed, increases in these exposure indicators are transitory and generally decrease to background levels within one year after the exposure. Laboratory studies have shown induction of cytochrome P450 systems occurs after exposure of fish to crude oil in water, sediment or food. Most of the PAH found in crude oil (dominantly 2- and 3-ring PAH) are not strong inducers of cytochrome P450. Exposure to the 4-ring chrysenes or the photooxidized products of the PAH may account for the cytochrome P450 responses in fish collected from oil-spill sites. The contribution of non-spill background PAH, particularly combustion-derived (pyrogenic) PAH, to bile FAC and cytochrome P450 system responses can be confounding and needs to be considered when evaluating oil spill effects. The ubiquity of pyrogenic PAH makes it important to fully characterize all sources of PAH, including PAH from natural resources, e.g. retene, in oil spill studies. In addition, such parameters as species, sex, age, ambient temperature and season need to be taken into account. While increases in fish bile FAC and cytochrome P450 system responses, can together, be sensitive general indicators of PAH exposure after an oil spill, there is little unequivocal evidence to suggest a linkage to

Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

Science.gov (United States)

Chen, Xi'en; Zhang, Yalin

2015-03-10

NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides. Copyright © 2015 Elsevier B.V. All rights reserved.

Probing cytochrome c in living mitochondria with surface-enhanced Raman spectroscopy

DEFF Research Database (Denmark)

Brazhe, Nadezda A.; Evlyukhin, Andrey B.; Goodilin, Eugene A.

2015-01-01

Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow...... due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living...... mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman...

Reprint of “Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus”

Energy Technology Data Exchange (ETDEWEB)

Borghese, Roberto, E-mail: roberto.borghese@unibo.it [Dept. of Pharmacy and Biotechnology, University of Bologna (Italy); Brucale, Marco [Institute for the Study of Nanostructured Materials (CNR-ISMN), Rome (Italy); Fortunato, Gianuario [Dept. of Pharmacy and Biotechnology, University of Bologna (Italy); Lanzi, Massimiliano [Dept. of Industrial Chemistry “Toso Montanari”, University of Bologna (Italy); Mezzi, Alessio [Institute for the Study of Nanostructured Materials (CNR-ISMN), Rome (Italy); Valle, Francesco; Cavallini, Massimiliano [Institute for the Study of Nanostructured Materials (CNR-ISMN), Bologna (Italy); Zannoni, Davide [Dept. of Pharmacy and Biotechnology, University of Bologna (Italy)

2017-02-15

Highlights: • Tellurite is reduced by R. capsulatus as cytosolic tellurium nanoprecipitates TeNPs. • Lawsone allows R. capsulatus to produce extracellular TeNPs. • Extracellular TeNPs production depends on the carbon source used for cells growth. • Both lawsone concentration and the incubation time determine the TeNPs size. • Extracellular TeNPs are coated with extracellular polymeric substances (EPS). - Abstract: The toxic oxyanion tellurite (TeO{sub 3}{sup 2−}) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te{sup 0} in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te{sup 0} nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1 mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600–700 nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te{sup 0} to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents.

Molecular genetic and molecular evolutionary studies on the bacteriochlorophyll synthesis genes of Rhodobacter capsulatus

Energy Technology Data Exchange (ETDEWEB)

Burke-Agueero, D.H.

1992-08-01

Rhodobacter capsulatus, purple bacterium capable of either aerobic or photosynthetic growth, has proven to be very useful in genetic studies of photosynthesis. Forty-four genes clustered together within a 46 kilobase region are required to establish photosynthetic ability in R. capsulatus. Approximately twenty of these genes are involved in bacteriochlorophyll synthesis of which eight bch'' genes are the subject of this thesis. Six of these genes were found to code for the two ring reductases. The first converts protochlorophyllide (PChlide) into a chlorin, the immediate precursor to chlorophyll a, and then into a bacteriochlorin. Each reductase is shown to be made up of three subunits. PChlide reductase is coded by the genes bchN, bchB, and bchL. Proteins with amino acid sequences markedly similar to those of bchN and bchL have been shown in other organisms to be required for chlorophyll synthesis; hence, their designation as chlN and chlB. A third chloroplast-encoded gene of heretofore unknown function shares amino acid identities with bchB and is probably the third subunit of the plant PChlide reductase. The bchA locus, which encodes the chlorin reductase, is found to be made up of three separate, translationally coupled genes, referred to as bchX, bchY, and bchZ. Amino acid similarities between bchX, bchL, and the nitrogenase reductase protein nifH suggest that all three classes of proteins share certain three-dimensional structural features, including elements that are central to the enzymatic mechanism of nifH. PChlide reductase and chlorin reductase are clearly derived from a common ancestor. Several lines of analysis suggests the ancestor of both enzyme systems reduced PChlide twice to produce bacteriochlorophyll supporting the concept bacteriochlorophyll as the ancestral reaction center pigment.

Molecular genetic and molecular evolutionary studies on the bacteriochlorophyll synthesis genes of Rhodobacter capsulatus

Energy Technology Data Exchange (ETDEWEB)

Burke-Agueero, Donald H. [Univ. of California, Berkeley, CA (United States)

1992-08-01

Rhodobacter capsulatus, purple bacterium capable of either aerobic or photosynthetic growth, has proven to be very useful in genetic studies of photosynthesis. Forty-four genes clustered together within a 46 kilobase region are required to establish photosynthetic ability in R. capsulatus. Approximately twenty of these genes are involved in bacteriochlorophyll synthesis of which eight ``bch`` genes are the subject of this thesis. Six of these genes were found to code for the two ring reductases. The first converts protochlorophyllide (PChlide) into a chlorin, the immediate precursor to chlorophyll a, and then into a bacteriochlorin. Each reductase is shown to be made up of three subunits. PChlide reductase is coded by the genes bchN, bchB, and bchL. Proteins with amino acid sequences markedly similar to those of bchN and bchL have been shown in other organisms to be required for chlorophyll synthesis; hence, their designation as chlN and chlB. A third chloroplast-encoded gene of heretofore unknown function shares amino acid identities with bchB and is probably the third subunit of the plant PChlide reductase. The bchA locus, which encodes the chlorin reductase, is found to be made up of three separate, translationally coupled genes, referred to as bchX, bchY, and bchZ. Amino acid similarities between bchX, bchL, and the nitrogenase reductase protein nifH suggest that all three classes of proteins share certain three-dimensional structural features, including elements that are central to the enzymatic mechanism of nifH. PChlide reductase and chlorin reductase are clearly derived from a common ancestor. Several lines of analysis suggests the ancestor of both enzyme systems reduced PChlide twice to produce bacteriochlorophyll supporting the concept bacteriochlorophyll as the ancestral reaction center pigment.

Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs.

Science.gov (United States)

Novak, David; Mojovic, Milos; Pavicevic, Aleksandra; Zatloukalova, Martina; Hernychova, Lenka; Bartosik, Martin; Vacek, Jan

2018-02-01

Cytochrome c (cyt c) is one of the most studied conjugated proteins due to its electron-transfer properties and ability to regulate the processes involved in homeostasis or apoptosis. Here we report an electrochemical strategy for investigating the electroactivity of cyt c and its analogs with a disrupted heme moiety, i.e. apocytochrome c (acyt c) and porphyrin cytochrome c (pcyt c). The electrochemical data are supplemented with low-temperature and spin-probe electron paramagnetic resonance (EPR) spectroscopy. The main contribution of this report is a complex evaluation of cyt c reduction and oxidation at the level of surface-localized amino acid residues and the heme moiety in a single electrochemical scan. The electrochemical pattern of cyt c is substantially different to both analogs acyt c and pcyt c, which could be applicable in further studies on the redox properties and structural stability of cytochromes and other hemeproteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

Czech Academy of Sciences Publication Activity Database

Koblížek, Michal; Shih, J. D.; Breitbart, S. I.; Ratcliffe, E. C.; Kolber, Z. S.; Hunter, C. N.; Niederman, R. A.

2005-01-01

Roč. 1706, - (2005), s. 220-231 ISSN 0006-3002 R&D Projects: GA ČR GP206/03/P079; GA MŠk LN00A141 Institutional research plan: CEZ:AV0Z50200510 Keywords : membrane development * light-harvesing complex * photosynthetic membrane Subject RIV: EE - Microbiology, Virology

The response of antioxidant systems in Nostoc sphaeroides against UV-B radiation and the protective effects of exogenous antioxidants

Science.gov (United States)

Wang, Gaohong; Hu, Chunxiang; Li, Dunhai; Zhang, Delu; Li, Xiaoyan; Chen, Kun; Liu, Yongding

UV radiation is one of many harmful factors found in space that are detrimental to organisms on earth in space exploration. In the present work, we examined the role of antioxidant system in Nostoc sphaeroides Kütz (Cyanobacterium) and the effects of exogenously applied antioxidant molecules on its photosynthetic rate under UV-B radiation. It was found that UV-B radiation promoted the activity of antioxidant system to protect photosystem II (PSII) and exogenously applied antioxidant: sodium nitroprusside (SNP) and N-acetylcysteine (NAC) had an obvious protection on PSII activity under UV-B radiation. The activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and content of MDA (malondialdehyde) and ASC (ascorbate) were improved by 0.5 mM and 1 mM SNP, but 0.1 mM SNP decreased the activity of antioxidant system. Addition of exogenous NAC decreased the activity of SOD, POD, CAT and the content MDA and ASC. In contrast, exogenously applied NAC increased GSH content. The results suggest that exogenous SNP and NAC may protect algae by different mechanisms: SNP may play double roles as both sources of reactive free radicals as well as ROS scavengers in mediating the protective role of PSII on algae under UV-B radiation. On the other hand, NAC functions as an antioxidant or precursor of glutathione, which could protect PSII directly from UV-B radiation.

c-Type cytochrome-dependent formation of U(IV nanoparticles by Shewanella oneidensis.

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Matthew J Marshall

2006-09-01

Full Text Available Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI complexes in situ, the biomolecular mechanisms of U(VI reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI and formation of extracellular UO(2 nanoparticles. In particular, the outer membrane (OM decaheme cytochrome MtrC (metal reduction, previously implicated in Mn(IV and Fe(III reduction, directly transferred electrons to U(VI. Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO(2 nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS. In wild-type cells, this UO(2-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO(2 nanoparticles with MtrC and OmcA (outer membrane cytochrome. This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO(2 nanoparticles. In the environment, such association of UO(2 nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O(2 or transport in soils and sediments.

Intestinal cytochromes P450 regulating the intestinal microbiota and its probiotic profile

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Eugenia Elefterios Venizelos Bezirtzoglou

2012-09-01

Full Text Available Cytochromes P450 (CYPs enzymes metabolize a large variety of xenobiotic substances. In this vein, a plethora of studies were conducted to investigate their role, as cytochromes are located in both liver and intestinal tissues. The P450 profile of the human intestine has not been fully characterized. Human intestine serves primarily as an absorptive organ for nutrients, although it has also the ability to metabolize drugs. CYPs are responsible for the majority of phase I drug metabolism reactions. CYP3A represents the major intestinal CYP (80% followed by CYP2C9. CYP1A is expressed at high level in the duodenum, together with less abundant levels of CYP2C8-10 and CYP2D6. Cytochromes present a genetic polymorphism intra- or interindividual and intra- or interethnic. Changes in the pharmacokinetic profile of the drug are associated with increased toxicity due to reduced metabolism, altered efficacy of the drug, increased production of toxic metabolites, and adverse drug interaction. The high metabolic capacity of the intestinal flora is due to its enormous pool of enzymes, which catalyzes reactions in phase I and phase II drug metabolism. Compromised intestinal barrier conditions, when rupture of the intestinal integrity occurs, could increase passive paracellular absorption. It is clear that high microbial intestinal charge following intestinal disturbances, ageing, environment, or food-associated ailments leads to the microbial metabolism of a drug before absorption. The effect of certain bacteria having a benefic action on the intestinal ecosystem has been largely discussed during the past few years by many authors. The aim of the probiotic approach is to repair the deficiencies in the gut flora and establish a protective effect. There is a tentative multifactorial association of the CYP (P450 cytochrome role in the different diseases states, environmental toxic effects or chemical exposures and nutritional status.

Identification of fraud (with pig stuffs) in chicken-processed meat through information of mitochondrial cytochrome b.

Science.gov (United States)

Yacoub, Haitham A; Sadek, Mahmoud A

2017-11-01

This study was conducted to find out the fraud in chicken-processed meat ingredients to protect consumers from commercial adulteration and authentication through a reliable way: direct amplification of conserved segment of cytochrome b gene of mitochondrial DNA, in addition, using species-specific primer assay for a certain cytochrome b. The results reported that chicken-processed meats were identified as a chicken meat based on amplification of conserved cytochrome b gene of mtDNA, while different fragments sizes were produced after the application of species-specific primer as follows: 227, 157, 274, 331, 389 and 439 bp for raw meat of chicken, goat, cattle, sheep, pig and horse, respectively. The results revealed that all chicken meat products are produced with 227 bp in size. While, an adulteration with pork stuffs was observed in some of the chicken meat products using a species-specific primer of cytochrome b gene, namely, chicken luncheon and chicken burger. This study represents a reliable technique that could be used to provide a promising solution for identifying the commercial adulteration and substitutions in processed meat in retail markets.

Heme exporter FLVCR1a regulates heme synthesis and degradation and controls activity of cytochromes P450.

Science.gov (United States)

Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

2014-05-01

The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Relative importance of driving force and electrostatic interactions in the reduction of multihaem cytochromes by small molecules.

Science.gov (United States)

Quintas, Pedro O; Cepeda, Andreia P; Borges, Nuno; Catarino, Teresa; Turner, David L

2013-06-01

Multihaem cytochromes are essential to the energetics of organisms capable of bioremediation and energy production. The haems in several of these cytochromes have been discriminated thermodynamically and their individual rates of reduction by small electron donors were characterized. The kinetic characterization of individual haems used the Marcus theory of electron transfer and assumed that the rates of reduction of each haem by sodium dithionite depend only on the driving force, while electrostatic interactions were neglected. To determine the relative importance of these factors in controlling the rates, we studied the effect of ionic strength on the redox potential and the rate of reduction by dithionite of native Methylophilus methylotrophus cytochrome c″ and three mutants at different pH values. We found that the main factor determining the rate is the driving force and that Marcus theory describes this satisfactorily. This validates the method of the simultaneous fitting of kinetic and thermodynamic data in multihaem cytochromes and opens the way for further investigation into the mechanisms of these proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

Cloning, expression and purification of cytochrome c{sub 6} from the brown alga Hizikia fusiformis and complete X-ray diffraction analysis of the structure

Energy Technology Data Exchange (ETDEWEB)

Akazaki, Hideharu [Bio-organic Chemistry Laboratory, Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa-shi, Kanagawa 252-8510 (Japan); Kawai, Fumihiro [Protein Design Laboratory, Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Chida, Hirotaka; Matsumoto, Yuichirou; Hirayama, Mao; Hoshikawa, Ken [Bio-organic Chemistry Laboratory, Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa-shi, Kanagawa 252-8510 (Japan); Unzai, Satoru [Protein Design Laboratory, Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Hakamata, Wataru; Nishio, Toshiyuki; Park, Sam-Yong; Oku, Tadatake, E-mail: oku@brs.nihon-u.ac.jp [Bio-organic Chemistry Laboratory, Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa-shi, Kanagawa 252-8510 (Japan)

2008-08-01

The crystal structure of cytochrome c{sub 6} from the brown alga H. fusiformis has been determined at 1.6 Å resolution. The amino-acid sequence and tertiary structure of H. fusiformis cytochrome c{sub 6} were very similar to those of red algal cytochrome c{sub 6} rather than those of green algal cytochrome c{sub 6}. The primary sequence of cytochrome c{sub 6} from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 Å resolution. The crystal belonged to the tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 84.58, c = 232.91 Å and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c{sub 6} were most similar to those of red algal cytochrome c{sub 6}. These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.

Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

Science.gov (United States)

Dodhia, Vikash Rajnikant; Fantuzzi, Andrea; Gilardi, Gianfranco

2006-10-01

The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

Oxygen consumption and cytochrome exidase activity of axolotl limbs muscle tissue in restoration of regenerative ability suprressed by X-irradiation

International Nuclear Information System (INIS)

Teplits, N.A.

1975-01-01

The rate of oxygen use and activity of cytochrome oxidase in a homogenate of mitochondria and nuclei of muscle tissue of axolotl limbs after suppression of their regenerative capability by x irradiation and their restoration was studied experimentally. With suppression of the regenative capability the use of oxygen was depressed. Cytochrome oxidase activity in the homogenate and mitochondria decreased compared to that of the intact limb, in the nuclei of muscle tissue it was the same or greater. With restoration of the regenerative capability of the limbs the respiration rate of the homogenate and the mitochondria increased, accompanied by increased cytochrome oxidase activity. In the nuclei the cytochrome oxidase activity did not change in the blastema stage and sharply decreased in the limb formation state. (E.T.)

Oxygen consumption and cytochrome exidase activity of axolotl limbs muscle tissue in restoration of regenerative ability suppressed by X-irradiation

Energy Technology Data Exchange (ETDEWEB)

Teplits, N A [AN SSSR, Moscow. Inst. Biologii Razvitiya

1975-01-01

The rate of oxygen use and activity of cytochrome oxidase in a homogenate of mitochondria and nuclei of muscle tissue of axolotl limbs after suppression of their regenerative capability by x irradiation and their restoration was studied experimentally. With suppression of the regenative capability the use of oxygen was depressed. Cytochrome oxidase activity in the homogenate and mitochondria decreased compared to that of the intact limb, in the nuclei of muscle tissue it was the same or greater. With restoration of the regenerative capability of the limbs the respiration rate of the homogenate and the mitochondria increased, accompanied by increased cytochrome oxidase activity. In the nuclei the cytochrome oxidase activity did not change in the blastema stage and sharply decreased in the limb formation state.

The effect of lycopene on the total cytochrome P450, CYP1A2 and CYP2E1

Directory of Open Access Journals (Sweden)

Melva Louisa

2009-12-01

Full Text Available Aim: Some carotenoids such as canthaxantin, astaxanthin and beta apo-8’-carotenal were reported to have modulatoryeffect on the cytochrome P450. The present study was conducted to investigate the effects of lycopene, a nonprovitamin A carotenoid, on microsomal cytochrome P450, CYP1A2 and CYP2E1.Methods: Total cytochrome P450 levels, CYP1A2 and CYP2E1-catalyzed reactions (acetanilide 4-hydroxylation and p-nitrophenol hydroxylation were studied in the liver microsomes of male Sprague Dawley rats. Microsomes were prepared using differential centrifugation combined with calcium aggregation method. Lycopene was orally administered in the dosages of 0, 25, 50 or 100 mg/kgBW/day for 14 days in a repeated fashion. Data were analyzed using ANOVA test.Results: Total cytochrome P450 level and acetanilide 4-hydroxylase activity were unaffected by any of the treatments. The CYP2E1 probe enzyme (p-nitrophenol hydroxylase was significantly reduced by repeated administration of 100mg/ kgBW/day lycopene (7.88 + 2.04 vs 12.26 + 2.77 n mol/min/mg prot.Conclusion: The present results suggest that lycopene does not affect the total cytochrome P450 or CYP1A2 activity but it inhibits the activity of CYP2E1 (p-nitrophenol hydroxylase in the rat. (Med J Indones 2009; 18: 233-8Keywords: lycopene, cytochrome P450, CYP1A2, CYP2E1

A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

Science.gov (United States)

Kiffel, L; Loeper, J; Homberg, J C; Leroux, J P

1989-02-28

1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

Cooperative use of cytochrome cd{sub 1} nitrite reductase and its redox partner cytochrome c{sub 552} to improve the selectivity of nitrite biosensing

Energy Technology Data Exchange (ETDEWEB)

Serra, A.S.; Jorge, S.R.; Silveira, C.M.; Moura, J.J.G. [REQUIMTE - Dept. de Quimica, CQFB, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Jubete, E.; Ochoteco, E.; Cabanero, G.; Grande, H. [CIDETEC - Centro de Tecnologias Electroquimicas, Parque Tecnologico de San Sebastian, Po Miramon, 196, 20009 Donostia - San Sebastian (Spain); Almeida, M.G., E-mail: mga@dq.fct.unl.pt [REQUIMTE - Dept. de Quimica, CQFB, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Escola Superior de Saude Egas Moniz, Monte de Caparica, 2829-511 Caparica (Portugal)

2011-05-05

In this work, a novel enzymatic biosensor for determination of nitrites constructed on an electrochemical transducing platform is proposed. The sensor is based on cytochrome-cd{sub 1} (cyt-cd{sub 1}) nitrite reductase from Marinobacter hydrocarbonoclasticus strain 617 as biological recognition element, and its putative physiological redox partner cytochrome-c{sub 552} (cyt-c{sub 552}), as electron mediator. The proteins were co-immobilized using a photopolymerizable polyvinyl alcohol (PVA) derivative, onto carbon paste screen printed electrodes (CPSPEs); the optimal modification conditions were 100 {mu}M cyt-cd{sub 1}/100 {mu}M cyt-c{sub 552} and 50% PVA, after a 48 h polymerization time. Electrochemical characterization of the mediator was carried out by cyclic voltammetry. The one-electron exchange between cyt-c{sub 552} and the working electrode is a quasi-reversible process, without mass transport limitations. The formal potential of the mediator is 254 {+-} 2 mV vs NHE and the intermolecular electron transfer rate constant between cytochromes c{sub 552} and cd{sub 1} is 9.9 x 10{sup 3} M{sup -1} s{sup -1}. The analytical parameters of the biosensor response to nitrite as assessed by amperometric measurements were: linear range from 10 to 200 {mu}M; detection and quantification limits of 7 and 24 {mu}M, respectively; sensitivity of 2.49 {+-} 0.08 A mol{sup -1} cm{sup 2} {mu}M{sup -1}. Catalytic profiles in the presence of possible interfering species were also investigated. The interference from competitive enzymatic reduction of dissolved oxygen could be overcome by tuning the cyclic voltammograms for faster sweep rates.

Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy

Science.gov (United States)

Forman, C. J.; Wang, N.; Yang, Z. Y.; Mowat, C. G.; Jarvis, S.; Durkan, C.; Barker, P. D.

2013-05-01

Amyloid fibres displaying cytochrome b562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias (<1.5 V) the fibres appeared as regions of low conductivity with no evidence of cytochrome mediated electron transfer. At a high bias, stable peaks in tunnelling current were observed for all three fibre species containing haem and one species of fibre that did not contain haem. In images of this kind, some of the current peaks were collinear and spaced around 10 nm apart over ranges longer than 100 nm, but background monomers complicate interpretation. Images of the third kind were rare (1 in 150 fibres); in these, fully conducting structures with the approximate dimensions of fibres were observed, suggesting the possibility of an intermittent conduction mechanism, for which a precedent exists in DNA. To test the conductivity, some fibres were immobilized with sputtered gold, and no evidence of conduction between the grains of gold was seen. In control experiments, a variation of monomeric cytochrome b562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid.

Effect of retinal impulse blockage on cytochrome oxidase-poor interpuffs in the macaque striate cortex: quantitative EM analysis of neurons.

Science.gov (United States)

Wong-Riley, M T; Trusk, T C; Kaboord, W; Huang, Z

1994-09-01

One of the hallmarks of the primate striate cortex is the presence of cytochrome oxidase-rich puffs in its supragranular layers. Neurons in puffs have been classified as type A, B, and C in ascending order of cytochrome oxidase content, with type C cells being the most vulnerable to retinal impulse blockade. The present study aimed at analysing cytochrome oxidase-poor interpuffs with reference to their metabolic cell types and the effect of intraretinal tetrodotoxin treatment. The same three metabolic types were found in interpuffs, except that type B and C neurons were smaller and less cytochrome oxidase-reactive in interpuffs than in puffs. Type A neurons had small perikarya, low levels of cytochrome oxidase, and received exclusively symmetric axosomatic synapses. The largest neurons were pyramidal, type B cells with moderate cytochrome oxidase activity and were also contacted exclusively by symmetric axosomatic synapses. Type C cells medium-sized with a rich supply of large, darkly reactive mitochondria and possessed all the characteristics of GABAergic neurons. They were the only cell type that received both symmetric and asymmetric axosomatic synapses. Two weeks of monocular tetrodotoxin blockade in adult monkeys caused all three major cell types in deprived interpuffs to suffer a significant downward shift in the size and cytochrome oxidase reactivity of their mitochondria, but the effects were more severe in type B and C neurons. In nondeprived interpuffs, all three cell types gained both in size and absolute number of mitochondria, and type A cells also had an elevated level of cytochrome oxidase, indicating that they might be functioning at a competitive advantage over cells in deprived columns. However, type B and C neurons showed a net loss of darkly reactive mitochondria, indicating that these cells became less active. Thus, mature interpuff neurons remained vulnerable to retinal impulse blockade and the metabolic capacity of these cells remains tightly

Cytochrome and Alternative Pathway Respiration in Tobacco (Effects of Salicylic Acid).

Science.gov (United States)

Rhoads, D. M.; McIntosh, L.

1993-11-01

In suspension cultures of NT1 tobacco (Nicotiana tabacum L. cv Bright Yellow) cells the cytochrome pathway capacity increased between d 3 and d 4 following subculturing and reached the highest level observed on d 7. The capacity decreased significantly by d 10 and was at the same level on d 14. Both alternative pathway capacity and the amount of the 35-kD alternative oxidase protein increased significantly between d 5 and d 6, reached the highest point observed on d 7, remained constant until d 10, and decreased by d 14. The highest capacities of the alternative and cytochrome pathways and the highest amount of the 35-kD protein were attained on the day that cell cultures reached a stationary phase of growth. Addition of salicylic acid to cell cultures on d 4 caused a significant increase in alternative pathway capacity and a dramatic accumulation of the 35-kD protein by 12 h. The alternative pathway capacity and the protein level reached the highest level observed by 16 h after salicylic acid addition, and the cytochrome pathway capacity was at about the same level at each time point. The accumulation of the 35-kD alternative oxidase protein was significantly decreased by addition of actinomycin D 1 h before salicylic acid and was blocked by addition of cycloheximide. These results indicate that de novo transcription and translation were necessary for salicylic acid to cause the maximum accumulation of the 35-kD protein.

Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase.

Science.gov (United States)

Lehninger, A L; Reynafarje, B; Costa, L

1985-01-01

The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.

HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity

Science.gov (United States)

Furge, Laura Lowe; Fletke, Kyle J.

2007-01-01

Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among…

Redox Thermodynamics of Cytochromes c Subjected to Urea Induced Unfolding

NARCIS (Netherlands)

Monari, S.; Ranieri, A.; Di Rocco, G.; van der Zwan, G.; Peressini, S.; Tavagnacco, C.; Millo, D.; Borsari, M.

2009-01-01

The thermodynamics of the electron transfer (ET) process for beef heart and yeast cytochromes c and the Lys72Ala/Lys73Ala/Lys79Ala mutant of the latter species subjected to progressive urea-induced unfolding was determined electrochemically. The results indicate the presence of at least three

Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy

International Nuclear Information System (INIS)

Forman, C J; Barker, P D; Wang, N; Durkan, C; Yang, Z Y; Mowat, C G; Jarvis, S

2013-01-01

Amyloid fibres displaying cytochrome b 562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias ( 562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid. (paper)

Genome mining in Sorangium cellulosum So ce56: identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450.

Science.gov (United States)

Ewen, Kerstin Maria; Hannemann, Frank; Khatri, Yogan; Perlova, Olena; Kappl, Reinhard; Krug, Daniel; Hüttermann, Jürgen; Müller, Rolf; Bernhardt, Rita

2009-10-16

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.

The cytochrome bd-type quinol oxidase is important for survival of Mycobacterium smegmatis under peroxide and antibiotic-induced stress.

NARCIS (Netherlands)

Lu, P.; Heineke, M.H.; Koul, A.; Andries, K.; Cook, G.M.; Lill, H.; van Spanning, R.J.M.; Bald, D.

2015-01-01

Targeting respiration and ATP synthesis has received strong interest as a new strategy for combatting drug-resistant Mycobacterium tuberculosis. Mycobacteria employ a respiratory chain terminating with two branches. One of the branches includes a cytochrome bc 1 complex and an aa 3 -type cytochrome

Contribution of Electrostatics to the Kinetics of Electron Transfer from NADH-Cytochrome b5 Reductase to Fe(III)-Cytochrome b5.

Science.gov (United States)

Kollipara, Sireesha; Tatireddy, Shivakishore; Pathirathne, Thusitha; Rathnayake, Lasantha K; Northrup, Scott H

2016-08-25

Brownian dynamics (BD) simulations provide here a theoretical atomic-level treatment of the reduction of human ferric cytochrome b5 (cyt b5) by NADH-cytochrome b5 reductaste (cyt b5r) and several of its mutants. BD is used to calculate the second-order rate constant of electron transfer (ET) between the proteins for direct correlation with experiments. Interestingly, the inclusion of electrostatic forces dramatically increases the reaction rate of the native proteins despite the overall negative charge of both proteins. The role played by electrostatic charge distribution in stabilizing the ET complexes and the role of mutations of several amino acid residues in stabilizing or destabilizing the complexes are analyzed. The complex with the shortest ET reaction distance (d = 6.58 Å) from rigid body BD is further subjected to 1 ns of molecular dynamics (MD) in a periodic box of TIP3P water to produce a more stable complex allowed by flexibility and with a shorter average reaction distance d = 6.02 Å. We predict a docking model in which the following ion-ion interactions are dominant (cyt b5r/cyt b5): Lys162-Heme O1D/Lys163-Asp64/Arg91-Heme O1A/Lys125-Asp70.

Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

Science.gov (United States)

Zahn, J A; Duncan, C; DiSpirito, A A

1994-01-01

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline. Images PMID:7928947

Prognostic Value of Cytochrome C and Cytokines in Acute Viral Encephalopathy

Directory of Open Access Journals (Sweden)

J Gordon Millichap

2006-06-01

Full Text Available Serum cytochrome c and cytokines were evaluated as prognostic predictors in 29 children (ages 9 mos to 9 yrs 11 mos with viral acute encephalopathies and multiple organ failure at Fukushima Medical University School of Medicine, Japan.

Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

Science.gov (United States)

Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

2013-01-01

Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems. Copyright © 2011 John Wiley & Sons, Ltd.

Key role of water in proton transfer at the Q(o)-site of the cytochrome bc(1) complex predicted by atomistic molecular dynamics simulations

DEFF Research Database (Denmark)

Postila, P. A.; Kaszuba, K.; Sarewicz, M.

2013-01-01

of the cyt bc(1) function have remained unclear especially regarding the substrate binding at the Q(o)-site. In this work we address this issue by performing extensive atomistic molecular dynamics simulations with the cyt bc(1) complex of Rhodobacter capsulatus embedded in a lipid bilayer. Based...... on the simulations we are able to show the atom-level binding modes of two substrate forms: quinol (QH(2)) and quinone (Q). The QH(2) binding at the Q(o)-site involves a coordinated water arrangement that produces an exceptionally close and stable interaction between the cyt b and iron sulfur protein subunits...

Identification of a c-Type Cytochrome Specific for Manganese Dioxide (MnO2) Reduction in Anaeromyxobacter dehalogenans Strain 2CP-C

Science.gov (United States)

Pfiffner, S. M.; Nissen, S.; Liu, X.; Chourey, K.; Vishnivetskaya, T. A.; Hettich, R.; Loeffler, F.

2014-12-01

Anaeromyxobacter dehalogenans is a metabolically versatile Deltaproteobacterium and conserves energy from the reduction of various electron acceptors, including insoluble MnO2 and ferric oxides/oxyhydroxides (FeOOH). The goal of this study was to identify c-type cytochromes involved in electron transfer to MnO2. The characterization of deletion mutants has revealed a number of c-type cytochromes involved in electron transfer to solid metal oxides in Shewanella spp. and Geobacter spp; however, a genetic system for Anaeromyxobacter is not available. The A. dehalogenans str. 2CP-C genome encodes 68 putative c-type cytochromes, which all lack functional assignments. To identify c-type cytochromes involved in electron transfer to solid MnO2, protein expression profiles of A. dehalogenans str. 2CP-C cells grown with acetate as electron donor and MnO2, ferric citrate, FeOOH, nitrate or fumarate as electron acceptors were compared. Whole cell proteomes were analyzed after trypsin proteolysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Distinct c-type cytochrome expression patterns were observed with cells grown with different electron acceptors. A. dehalogenans str. 2CP-C grown with MnO2 expressed 25 out of the 68 c-type cytochromes encoded on the genome. The c-type cytochrome Adeh_1278 was only expressed in strain 2CP-C grown with MnO2. Reverse transcription PCR confirmed that the Adeh_1278 gene was transcribed in MnO2-grown cells but not in cells grown with other terminal electron acceptors. The expression of the Adeh_1278 gene correlated with Mn(IV) reduction activity. Adeh_1278 has three heme binding motifs and is predicted to be located in the periplasm. The identification of Adeh_1278 as a protein uniquely expressed when MnO2 serves as electron acceptor suggests its utility as a biomarker for MnO2 reduction. This example demonstrates the value of the LC-MS/MS approach for identifying specific proteins of interest and making functional assignments

Interplay between cytochrome c and gibberellins during Arabidopsis vegetative development

Czech Academy of Sciences Publication Activity Database

Racca, S.; Welchen, E.; Gras, D. E.; Tarkowská, Danuše; Turečková, Veronika; Maurino, V. G.; Gonzalez, D. H.

2018-01-01

Roč. 94, č. 1 (2018), s. 105-121 ISSN 0960-7412 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Arabidopsis thaliana * cytochrome c * DELLA protein * gibberellin * mitochondrion Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 5.901, year: 2016

Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

Science.gov (United States)

Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

2014-01-01

Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

Prediction of cytochrome P450 mediated metabolism

DEFF Research Database (Denmark)

Olsen, Lars; Oostenbrink, Chris; Jørgensen, Flemming Steen

2015-01-01

Cytochrome P450 enzymes (CYPs) form one of the most important enzyme families involved in the metabolism of xenobiotics. CYPs comprise many isoforms, which catalyze a wide variety of reactions, and potentially, a large number of different metabolites can be formed. However, it is often hard...... to rationalize what metabolites these enzymes generate. In recent years, many different in silico approaches have been developed to predict binding or regioselective product formation for the different CYP isoforms. These comprise ligand-based methods that are trained on experimental CYP data and structure...

Cytochrome P450-mediated metabolism of tumour promoters modifies the inhibition of intercellular communication: a modified assay for tumour promotion

DEFF Research Database (Denmark)

Vang, Ole; Wallin, H.; Doehmer, J.

1993-01-01

The role of metabolism of tumour promoters on the inhibition of intercellular communication was investigated in a modified V79 metabolic cooperation system. V79 cells, which stably express different rat cytochrome P450 enzymes (CYP1A1, CYP1A2 or CYP2B1), were used in the metabolic cooperation assay...... B1 and 4-nitrobiphenyl, did not inhibit metabolic cooperation in either V79 cells expressing or cells not expressing cytochrome P450. We conclude that cytochrome P450-associated metabolism plays an important role in the inhibition of gap junctional intercellular communication of some tumour...... promoters. The modified metabolic cooperation assay presented here is valuable for detecting some inhibitory chemicals which have been 'false negative' in previous assays for gap junctional intercellular communication. The assay also discloses that cytochrome P450 metabolism alters intercellular...

Redox active molecules cytochrome c and vitamin C enhance heme-enzyme peroxidations by serving as non-specific agents for redox relay

International Nuclear Information System (INIS)

Gade, Sudeep Kumar; Bhattacharya, Subarna; Manoj, Kelath Murali

2012-01-01

Highlights: ► At low concentrations, cytochrome c/vitamin C do not catalyze peroxidations. ► But low levels of cytochrome c/vitamin C enhance diverse heme peroxidase activities. ► Enhancement positively correlates to the concentration of peroxide in reaction. ► Reducible additives serve as non-specific agents for redox relay in the system. ► Insight into electron transfer processes in routine and oxidative-stress states. -- Abstract: We report that incorporation of very low concentrations of redox protein cytochrome c and redox active small molecule vitamin C impacted the outcome of one-electron oxidations mediated by structurally distinct plant/fungal heme peroxidases. Evidence suggests that cytochrome c and vitamin C function as a redox relay for diffusible reduced oxygen species in the reaction system, without invoking specific or affinity-based molecular interactions for electron transfers. The findings provide novel perspectives to understanding – (1) the promiscuous role of cytochrome b 5 in the metabolism mediated by liver microsomal xenobiotic metabolizing systems and (2) the roles of antioxidant molecules in affording relief from oxidative stress.

An extensive deletion causing overproduction of yeast iso-2-cytochrome c

International Nuclear Information System (INIS)

McKnight, G.L.; Cardillo, T.S.; Sherman, F.

1981-01-01

CYC7-H3 is a cis-dominant regulatory mutation that causes a 20-fold overproduction of yeast iso-2-cytochrome c. The CYC7-H3 mutation is an approximately 5 kb deletion with one breakpoint located in the 5' noncoding region of the CYC7 gene, approximately 200 base from the ATG initiation codon. The deletion apparently fuses a new regulatory region to the structural portion of the CYC7 locus. The CYC7-H3 deletion encompasses the RAD23 locus, which controls UV sensitivity and the ANP1 locus, which controls osmotic sensitivity. The gene cluster CYC7-RAD23-ANP1 displays striking similarity to the gene cluster CYC1-OSM1-RAD7, which controls, respectively, iso-1-cytochrome c, osmotic sensitivity and UV sensitivity. We suggest that these gene clusters are related by an ancient transpositional event

A theoretical study on the metabolic activation of paracetamol by cytochrome P-450 : indications for a uniform oxidation mechanism

NARCIS (Netherlands)

Koymans, L.; Lenthe, J.H.; Van de Straat, R; Donné-Op den Kelder, G M; Vermeulen, N P

1989-01-01

The cytochrome P-450 mediated activation of paracetamol (PAR) to the reactive electrophilic intermediate N-acetyl-p-benzoquinone imine (NAPQI) has been studied by use of SV 6-31G ab initio energy calculations and spin distributions. A simplified model for cytochrome P-450 has been used by

Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

Science.gov (United States)

Hiesel, Rudolf; Brennicke, Axel

1983-01-01

The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

Nitrate as a probe of cytochrome c surface: crystallographic identification of crucial "hot spots" for protein-protein recognition.

Science.gov (United States)

De March, Matteo; Demitri, Nicola; De Zorzi, Rita; Casini, Angela; Gabbiani, Chiara; Guerri, Annalisa; Messori, Luigi; Geremia, Silvano

2014-06-01

The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive macromolecular complexes and of their breakage following the electron transfer process, the X-ray structures of horse heart ferri-cytochrome c (trigonal form) and ferro-cytochrome c (monoclinic form) were obtained using nitrate ions both as a crystallizing agent and an anionic probe for mapping the electrostatic surface changes. Both crystal forms contain three protein molecules in the asymmetric unit. In addition, a total of 21.5 and 18 crystallographically independent nitrate ions were identified for the trigonal and monoclinic forms, respectively. By matching all the six crystallographically independent protein molecules, 26 different anion-protein interaction sites were identified on the surfaces of cytochrome c, 10 of which were found in both forms, 8 present only in the oxidized and 8 only in the reduced form. The structural analysis of the electron transfer complexes, based on this new information, suggests a specific exit strategy for cytochrome c after formation of productive protein-protein complexes: a directional sliding mechanism for the electron shuttle on the surface of the redox partner is proposed to take place after the electron transfer process has occurred. Copyright © 2014 Elsevier Inc. All rights reserved.

Certain tryptophan photoproducts are inhibitors of cytochrome P450-dependent mutagenicity

International Nuclear Information System (INIS)

Rannug, U.; Agurell, E.; Cederberg, H.; Rannug, A.

1992-01-01

Two photoproducts, derived from UV-irradiation of the amino acid L-tryptophan and with high Ah (TCDD) receptor binding affinity, were tested for genotoxic and antimutagenic effects. The two indolo[3,2-b]carbazole derivatives, with the molecular weights of 284 and 312, respectively, were tested in Saccharomyces cerevisiae strain D7 for mitotic gene conversion and reverse mutation and in strain RS112 for sister chromatid conversion and gene conversion. No significant (P > 0.05) genotoxic effects were found in strain D7, while strain RS112 showed a small but significant increase in the frequency of sister chromatid conversions. In Chinese hamster ovary (CHO) cells the two compounds induced a statistically significant but less than twofold increase in the frequency of sister chromatid exchanges (SCE). No mutations were detected when the compounds were tested in Salmonella tphimurium strains TA98 and TA100. However, both 284 and 312 acted as antimutagens on strain TA100+S9 in the presence of benzo(a)pyrene. The decrease in mutagenicity by the most potent compound 284 was 20 revertants/nmol. This effect could be explained by an inhibitory effect on the cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity as seen in rat hepatocytes. The two compounds were also tested with hamster cells expressing rat cytochrome P-4501A1. The results support the conclusion that this cytochrome P-450 isozyme is inhibited by the tryptophan photoproducts. Similar results were also seen with two other high affinity Ah receptor ligands the quinazolinocarboline alkaloids rutaecapine and dehydrorutaecarpine. 20 refs., 3 figs., 4 tabs

Comparison of basal and induced cytochromes P450 in 6 species of waterfowl

Science.gov (United States)

Melancon, M.J.; Rattner, B.A.; Hoffman, D.J.; Beeman, D.; Day, D.; Custer, T.

1999-01-01

Cytochrome P450-associated monooxygenase activities were measured in control and prototype inducer-treated mallard duck, black duck, wood duck, lesser scaup, Canada goose and mute swan. Ages of the birds ranged from pipping embryos (that were treated approximately 3 days before pipping) to adults. Three or more of the following hepatic microsomal monooxygenases were assayed in each species: Benzyloxyresorufin-O-dealkylase (BROD), Ethoxyresorufin-O-dealkylase (EROD), methoxyresorufin-O-dealkylase (MROD), and pentoxyresorufin-O-dealkylase (PROD). Baseline activities differed between species, but because of differences in ages, sources of the eggs or birds, and diets, these cannot be viewed as absolute differences. The cytochrome P450 inducers utilized were beta-naphthoflavone (BNF), 3-methylcholanthrene (3MC) and phenobarbital (PB). In general, there was little response to PB; only lesser scaup were induced to greater than three times control level and most species were well under this. Responses to BNF and 3MC occurred in each species studied, but differed in which of the monooxygenases was most induced (absolute values and ratios to control values) and in relative induction between species. BROD frequently had an induction ratio EROD. Overall, lesser scaup were the most responsive, canada geese the least responsive, and the other species intermediate in responsiveness to the cytochrome P450 inducers studied.

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

Energy Technology Data Exchange (ETDEWEB)

Chinchilla, Diana, E-mail: Diana_Chinchilla@yahoo.com; Kilheeney, Heather, E-mail: raindropszoo@yahoo.com; Vitello, Lidia B., E-mail: lvitello@niu.edu; Erman, James E., E-mail: jerman@niu.edu

2014-01-03

Highlights: •Cytochrome c peroxidase (CcP) binds acrylonitrile in a pH-independent fashion. •The spectrum of the CcP/acrylonitrile complex is that of a 6c–ls ferric heme. •The acrylonitrile/CcP complex has a K{sub D} value of 1.1 ± 0.2 M. •CcP compound I oxidizes acrylonitrile with a maximum turnover rate of 0.61 min{sup −1}. -- Abstract: Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M{sup −1} s{sup −1} and 0.34 ± 0.15 s{sup −1}, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min{sup −1} at pH 6.0.

CYTOCHROME P450-DEPENDENT METABOLISM OF TRICHLOROETHYLENE IN THE RAT KIDNEY

Science.gov (United States)

The metabolism of trichloroethylene (Tri) by cytochrome P450 (P450) was studied in microsomes from liver and kidney homogenates and from isolated renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. Chloral hydrate (CH) was the only metabolite con...

INTERACTION OF AROMATIC CYTOKININS WITH HUMAN LIVER MICROSOMAL CYTOCHROMES P450

Czech Academy of Sciences Publication Activity Database

Anzenbacherová, E.; Janalík, J.; Popa, Igor; Strnad, Miroslav; Anzenbacher, P.

2005-01-01

Roč. 149, č. 2 (2005), s. 349-351 ISSN 1213-8118 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cytokinins * Cyclin dependent kinase inhibitor * Cytochrome P450 Subject RIV: CE - Biochemistry http://publib.upol.cz/~obd/fulltext/Biomed/2005/2/349.pdf

The interaction of representative members from two classes of antimycotics--the azoles and the allylamines--with cytochromes P-450 in steroidogenic tissues and liver.

Science.gov (United States)

Schuster, I

1985-06-01

Spectrophotometric studies with ketoconazole, clotrimazole and miconazole show strong type-II interactions with several cytochromes P-450, particularly (Ks greater than 10(7)M-1; pH7.4; 25 degrees C) with the 11 beta-hydroxylase of adrenal mitochondria, with the 17 alpha/20 lyase of testis microsomes and with some forms of cytochromes P-450 of liver. A tight binding of the azoles also occurs to the reduced cytochromes, giving rise to an impeded CO binding to the haem iron. The binding of the azoles to 11 beta-hydroxylase and 17 alpha/20 lyase is much tighter than the binding of endogenous substrates, and consequently inhibition of steroidogenesis will occur at these sites. The metabolism of xenobiotic substrates by the cytochromes P-450 of liver will also be severely impeded. In contrast, the allylamines naftifine and SF 86-327 are type-I substrates for a small portion of cytochromes P-450 of liver microsomes only and there is no spectral evidence for binding to the cytochromes P-450 involved in steroid biosynthesis.

The mechanism of electron gating in proton pumping cytochrome c oxidase: the effect of pH and temperature on internal electron transfer.

Science.gov (United States)

Brzezinski, P; Malmström, B G

1987-10-29

Electron-transfer reactions following flash photolysis of the mixed-valence cytochrome oxidase-CO complex have been measured at 445, 598 and 830 nm between pH 5.2 and 9.0 in the temperature range of 0-25 degrees C. There is a rapid electron transfer from the cytochrome a3-CuB pair to CuA (time constant: 14200 s-1), which is followed by a slower electron transfer to cytochrome a. Both the rate and the amplitude of the rapid phase are independent of pH, and the rate in the direction from CuA to cytochrome a3-CuB is practically independent of temperature. The second phase depends strongly on pH due to the titration of an acid-base group with pKa = 7.6. The equilibrium at pH 7.4 corresponds to reduction potentials of 225 and 345 mV for cytochrome a and a3, respectively, from which it is concluded that the enzyme is in a different conformation compared to the fully oxidized form. The results have been used to suggest a series of reaction steps in a cycle of the oxidase as a proton pump. Application of the electron-transfer theory to the temperature-dependence data suggests a mechanism for electron gating in the pump. Reduction of both cytochrome a and CuA leads to a conformational change, which changes the structure of cytochrome a3-CuB in such a way that the reorganizational barrier for electron transfer is removed and the driving force is increased.

Synchronous Measurement of Ultrafast Anisotropy Decay of the B850 in Bacterial LH2 Complex

International Nuclear Information System (INIS)

Wang Yun-Peng; Du Lu-Chao; Zhu Gang-Bei; Wang Zhuan; Weng Yu-Xiang

2015-01-01

Ultrafast anisotropic decay is a prominent parameter revealing ultrafast energy and electron transfer; however, it is difficult to be determined reliably owing to the requirement of a simultaneous availability of the parallel and perpendicular polarized decay kinetics. Nowadays, any measurement of anisotropic decay is a kind of approach to the exact simultaneity. Here we report a novel method for a synchronous ultrafast anisotropy decay measurement, which can well determine the anisotropy, even at a very early time, as the rising phase of the excitation laser pulse. The anisotropic decay of the B850 in bacterial light harvesting antenna complex LH2 of Rhodobacter sphaeroides in solution at room temperature with coherent excitation is detected by this method, which shows a polarization response time of 30 fs, and the energy transfer from the initial excitation to the bacteriochlorophylls in B850 ring takes about 70 fs. The anisotropic decay that is probed at the red side of the absorption spectrum, such as 880 nm, has an initial value of 0.4, corresponding to simulated emission, while the blue side with an anisotropy of 0.1 contributes to the ground-state bleaching. Our results show that the coherent excitation covering the whole ring might not be realized owing to the symmetry breaking of LH2: from C_9 symmetry in membrane to C_2 symmetry in solution. (atomic and molecular physics)

Synchronous Measurement of Ultrafast Anisotropy Decay of the B850 in Bacterial LH2 Complex

Science.gov (United States)

Wang, Yun-Peng; Du, Lu-Chao; Zhu, Gang-Bei; Wang, Zhuan; Weng, Yu-Xiang

2015-02-01

Ultrafast anisotropic decay is a prominent parameter revealing ultrafast energy and electron transfer; however, it is difficult to be determined reliably owing to the requirement of a simultaneous availability of the parallel and perpendicular polarized decay kinetics. Nowadays, any measurement of anisotropic decay is a kind of approach to the exact simultaneity. Here we report a novel method for a synchronous ultrafast anisotropy decay measurement, which can well determine the anisotropy, even at a very early time, as the rising phase of the excitation laser pulse. The anisotropic decay of the B850 in bacterial light harvesting antenna complex LH2 of Rhodobacter sphaeroides in solution at room temperature with coherent excitation is detected by this method, which shows a polarization response time of 30 fs, and the energy transfer from the initial excitation to the bacteriochlorophylls in B850 ring takes about 70 fs. The anisotropic decay that is probed at the red side of the absorption spectrum, such as 880 nm, has an initial value of 0.4, corresponding to simulated emission, while the blue side with an anisotropy of 0.1 contributes to the ground-state bleaching. Our results show that the coherent excitation covering the whole ring might not be realized owing to the symmetry breaking of LH2: from C9 symmetry in membrane to C2 symmetry in solution.

A comparative spectroscopic and kinetic study of photoexcitations in detergent-isolated and membrane-embedded LH2 light-harvesting complexes.

Science.gov (United States)

Freiberg, Arvi; Rätsep, Margus; Timpmann, Kõu

2012-08-01

Integral membrane proteins constitute more than third of the total number of proteins present in organisms. Solubilization with mild detergents is a common technique to study the structure, dynamics, and catalytic activity of these proteins in purified form. However beneficial the use of detergents may be for protein extraction, the membrane proteins are often denatured by detergent solubilization as a result of native lipid membrane interactions having been modified. Versatile investigations of the properties of membrane-embedded and detergent-isolated proteins are, therefore, required to evaluate the consequences of the solubilization procedure. Herein, the spectroscopic and kinetic fingerprints have been established that distinguish excitons in individual detergent-solubilized LH2 light-harvesting pigment-protein complexes from them in the membrane-embedded complexes of purple photosynthetic bacteria Rhodobacter sphaeroides. A wide arsenal of spectroscopic techniques in visible optical range that include conventional broadband absorption-fluorescence, fluorescence anisotropy excitation, spectrally selective hole burning and fluorescence line-narrowing, and transient absorption-fluorescence have been applied over broad temperature range between physiological and liquid He temperatures. Significant changes in energetics and dynamics of the antenna excitons upon self-assembly of the proteins into intracytoplasmic membranes are observed, analyzed, and discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. Copyright © 2011. Published by Elsevier B.V.

Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars.

Science.gov (United States)

Jose, Joachim; von Schwichow, Steffen

2004-04-02

Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).

RpoH2 sigma factor controls the photooxidative stress response in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7.

Science.gov (United States)

Kumar, Santosh; Rai, Ashutosh Kumar; Mishra, Mukti Nath; Shukla, Mansi; Singh, Pradhyumna Kumar; Tripathi, Anil Kumar

2012-12-01

Bacteria belonging to the Alphaproteobacteria normally harbour multiple copies of the heat shock sigma factor (known as σ(32), σ(H) or RpoH). Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours five copies of rpoH genes, one of which is an rpoH2 homologue. The genes around the rpoH2 locus in A. brasilense show synteny with that found in rhizobia. The rpoH2 of A. brasilense was able to complement the temperature-sensitive phenotype of the Escherichia coli rpoH mutant. Inactivation of rpoH2 in A. brasilense results in increased sensitivity to methylene blue and to triphenyl tetrazolium chloride (TTC). Exposure of A. brasilense to TTC and the singlet oxygen-generating agent methylene blue induced several-fold higher expression of rpoH2. Comparison of the proteome of A. brasilense with its rpoH2 deletion mutant and with an A. brasilense strain overexpressing rpoH2 revealed chaperone GroEL, elongation factors (Ef-Tu and EF-G), peptidyl prolyl isomerase, and peptide methionine sulfoxide reductase as the major proteins whose expression was controlled by RpoH2. Here, we show that the RpoH2 sigma factor-controlled photooxidative stress response in A. brasilense is similar to that in the photosynthetic bacterium Rhodobacter sphaeroides, but that RpoH2 is not involved in the detoxification of methylglyoxal in A. brasilense.

Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c(4) by high-cell-density fed-batch cultivation of Pseudomonas putida

DEFF Research Database (Denmark)

Thuesen, Marianne Hallberg; Nørgaard, Allan; Hansen, Anne Merete

2003-01-01

The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein...

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans

International Nuclear Information System (INIS)

Peschek, G.A.; Wastyn, M.; Trnka, M.; Molitor, V.; Fry, I.V.; Packer, L.

1989-01-01

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [ 35 S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min -1 (mg of protein) -1 , respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa 3 -type enzyme from the properties of its redox-active and EDTA-resistant Cu 2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa 3 -type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa 3 -type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme

Preferential hydroxylation over epoxidation catalysis by a horseradish peroxidase mutant: a cytochrome P450 mimic.

Science.gov (United States)

de Visser, Sam P

2007-10-25

Density functional theory calculations are presented on the catalytic properties of a horseradish peroxidase mutant whereby the axial nitrogen atom is replaced by phosphorus. This mutant has never been studied experimentally and only one theoretical report on this system is known (de Visser, S. P. J. Phys. Chem. B 2006, 110, 20759-20761). Thus, a one-atom substitution in horseradish peroxidase changes the properties of the catalytic center of the enzyme to more cytochrome P450-type qualities. In particular, the phosphorus-substituted horseradish peroxidase mutant reacts with substrates via a unique reactivity pattern, whereby alkanes are regioselectively hydroxylated even in the presence of a double bond. Reaction barriers of propene epoxidation and hydroxylation are almost identical to ones observed for a cytochrome P450 catalyst and significantly higher than those obtained for a horseradish peroxidase catalyst. It is shown that the regioselectivity difference is entropy and thermally driven and that the electron-transfer processes that occur during the reaction mechanism follow cytochrome P450-type patterns in the hydroxylation reaction.

Covalently Immobilised Cytochrome C Imaged by In Situ Scanning Tunnelling Microscopy

DEFF Research Database (Denmark)

Andersen, Jens Enevold Thaulov; Olesen, Klaus G.; Danilov, Alexey I.

1997-01-01

In situ scanning tunnelling microscopy (STM) imaging of cytochrome c (cyt c) on polycrystalline Pt surfaces and on Au(lll) was achieved first by covalent immobilisation of 3-aminopropyltriethoxysilane (3-APTS) brought to react with oxide present on the Pt surfaces. Covalently bound 3-APTS forms...

The Use of Cytochrome b Gene as a Specific Marker of the Rat Meat (Rattus norvegicus on Meat and Meat Products

Directory of Open Access Journals (Sweden)

C. Sumantri

2012-04-01

Full Text Available Falsification of the origin of livestock meat and its processed with rat meat is a problem that must be overcome to ensure food safety. One way that is often used to detect forgeries by using cytochrome b gene as a marker. The purpose of this study was to create a specific primer derived from cytochrome b sequences in rat (Rattus norvegicus as the DNA marker to detect any contamination of rat meat on fresh livestock meat and its processed meat products. Meatballs were made from beef meat with the addition of rat 1%-25%, and the meatballs were obtained from traditional markets. DNA extraction was conducted from seven species (goat, chicken, cattle, sheep, pig, horse, and rat by using phenol-chloroform. The highest success rate in detecting the presence of rat meat in a mixture of beef meatballs at concentration of 15% was 100%. The specific fragment of cytochrome b gene in R. norvegicus has no similarity with the cytochrome b gene from six other species, so it can be used as molecular markers to detect the presence of rat meat contamination in the processed of meat products. Amplified fragment length for goats, chickens, cattle, sheep, pigs, horses, and rats 157, 227, 274, 331, 398, 439 and 603 bp respectively. The amplification of cytochrome b gene in seven species of animals with different fragment length indicated the specificity of cytochrome b gene sequences among species.

Redox active molecules cytochrome c and vitamin C enhance heme-enzyme peroxidations by serving as non-specific agents for redox relay

Energy Technology Data Exchange (ETDEWEB)

Gade, Sudeep Kumar; Bhattacharya, Subarna [Heme and Flavo Proteins Laboratory, 204, Center for Biomedical Research, VIT University, Vellore, Tamil Nadu 632014 (India); Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com [Heme and Flavo Proteins Laboratory, 204, Center for Biomedical Research, VIT University, Vellore, Tamil Nadu 632014 (India)

2012-03-09

Highlights: Black-Right-Pointing-Pointer At low concentrations, cytochrome c/vitamin C do not catalyze peroxidations. Black-Right-Pointing-Pointer But low levels of cytochrome c/vitamin C enhance diverse heme peroxidase activities. Black-Right-Pointing-Pointer Enhancement positively correlates to the concentration of peroxide in reaction. Black-Right-Pointing-Pointer Reducible additives serve as non-specific agents for redox relay in the system. Black-Right-Pointing-Pointer Insight into electron transfer processes in routine and oxidative-stress states. -- Abstract: We report that incorporation of very low concentrations of redox protein cytochrome c and redox active small molecule vitamin C impacted the outcome of one-electron oxidations mediated by structurally distinct plant/fungal heme peroxidases. Evidence suggests that cytochrome c and vitamin C function as a redox relay for diffusible reduced oxygen species in the reaction system, without invoking specific or affinity-based molecular interactions for electron transfers. The findings provide novel perspectives to understanding - (1) the promiscuous role of cytochrome b{sub 5} in the metabolism mediated by liver microsomal xenobiotic metabolizing systems and (2) the roles of antioxidant molecules in affording relief from oxidative stress.

Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase

International Nuclear Information System (INIS)

Aigrain, Louise; Pompon, Denis; Truan, Gilles; Moréra, Solange

2009-01-01

A 2.5 Å resolution data set was collected from a crystal of a soluble chimeric form of NADPH-cytochrome P450 reductase (CPR) produced using a fusion gene composed of the yeast FMN and the human FAD domains. The chimeric protein was crystallized in a modified conformation compared with the previously solved structures. NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures

Humanlike substitutions to Ω-loop D of yeast iso-1-cytochrome c only modestly affect dynamics and peroxidase activity.

Science.gov (United States)

Lei, Haotian; Bowler, Bruce E

2018-06-01

Structural studies of yeast iso-1-cytochrome c (L.J. McClelland, T.-C. Mou, M.E. Jeakins-Cooley, S.R. Sprang, B.E. Bowler, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 6648-6653) show that modest movement of Ω-loop D (residues 70-85, average RMSD versus the native structure: 0.81 Å) permits loss of Met80-heme ligation creating an available coordination site to catalyze the peroxidase activity mediated by cytochrome c early in apoptosis. However, Ala81 and Gly83 move significantly (RMSDs of 2.18 and 1.26 Å, respectively). Ala81 and Gly83 evolve to Ile and Val, respectively, in human cytochrome c and peroxidase activity decreases 25-fold relative to the yeast protein at pH 7. To test the hypothesis that these residues evolved to restrict the peroxidase activity of cytochrome c, A81I and G83V variants of yeast iso-1-cytochrome c were prepared. For both variants, the apparent pK a of the alkaline transition increases by 0.2 to 0.3 relative to the wild type (WT) protein and the rate of opening the heme crevice is slowed. The cooperativity of acid unfolding is decreased for the G83V variant. At pH 7 and 8, the catalytic rate constant, k cat , for the peroxidase activity of both variants decreases relative to WT, consistent with the effects on alkaline isomerization. Below pH 7, the loss in the cooperativity of acid unfolding causes k cat for peroxidase activity to increase for the G83V variant relative to WT. Neither variant decreases k cat to the level of the human protein, indicating that other residues also contribute to the low peroxidase activity of human cytochrome c. Copyright © 2018 Elsevier Inc. All rights reserved.

Interaction of rocuronium with human liver cytochromes P450

OpenAIRE

Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

2015-01-01

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver micro...

The catalytic function of cytochrome P450 is entwined with its membrane-bound nature [version 1; referees: 4 approved

Directory of Open Access Journals (Sweden)

Carlo Barnaba

2017-05-01

Full Text Available Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts—cytochrome P450-reductase and cytochrome b5—are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.

Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.

Science.gov (United States)

Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R; Battaile, Kevin P; Lovell, Scott; Benson, David R; Zhu, Hao

2010-09-24

NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b(5) reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His(89) and His(112), consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b(5) family shown to have such a heme environment. Like other b(5) family members, Ncb5or-b(5) has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b(5) differs from Cyb5A with respect to location of the second heme ligand (His(112)) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b(5)R to Ncb5or-b(5) is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b(5) and b(5)R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b(5)R domains suggest that the CS domain facilitates docking of the b(5) and b(5)R domains. Trp(114) is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b(5)R domain to the b(5) domain.

Patterning of electrically conductive poly(aniline-co-aniline sulfonic acid) and its application in the immobilization of cytochrome c

International Nuclear Information System (INIS)

Oh, Se Young; Oh, Il Soo; Choi, Jeong-Woo

2004-01-01

We have synthesized poly(aniline-co-aniline sulfonic acid) and then investigated the feasibility of application as a specific and electrically conductive binding template for biomolecules. Poly(aniline-co-aniline sulfonic acid)s were prepared by oxidation polymerization of aniline and aniline sulfonic acid under various ratios. A fine pattern of the conducting copolyaniline was obtained by using a deep UV lithographic technique. Cytochrome c was immobilized onto the photochemically patterned conducting copolyaniline with a self-assembly method. Physical and electrochemical properties of the self-assembled cytochrome c monolayer were studied from atomic force microscopy and cyclic voltammetry. The self-assembled cytochrome c monolayer immobilized onto the copolyaniline with a high electrical conductivity showed a high electrochemical activity

Manipulating the Energetics and Rates of Electron Transfer in Rhodobacter capsulatus Reaction Centers with Asymmetric Pigment Content

Energy Technology Data Exchange (ETDEWEB)

Faries, Kaitlyn M. [Department; Dylla, Nicholas P. [Biosciences Division, Argonne National Laboratory, Lemont, Illinois 60439, United States; Hanson, Deborah K. [Biosciences Division, Argonne National Laboratory, Lemont, Illinois 60439, United States; Holten, Dewey [Department; Laible, Philip D. [Biosciences Division, Argonne National Laboratory, Lemont, Illinois 60439, United States; Kirmaier, Christine [Department

2017-07-17

Seemingly redundant parallel pathways for electron transfer (ET), composed of identical sets of cofactors, are a cornerstone feature of photosynthetic reaction centers (RCs) involved in light-energy conversion. In native bacterial RCs, both A and B branches house one bacteriochlorophyll (BChl) and one bacteriopheophytin (BPh), but the A branch is used exclusively. Described herein are the results-obtained for two Rhodobacter capsulatus RCs with an unnaturally high degree of cofactor asymmetry, two BPh on the RC's B side and two BChl on the A side. These pigment changes derive, respectively, from the His(M180)Leu mutation [a BPh ((Phi(B)) replaces the B-side BChl (BB)], and the Leu(M212)His mutation [a BChl (beta(A))) replaces the A-side BPh (H-A)]. Additionally, Tyr(M208)Phe was employed to disfavor ET to the A branch; in one mutant, Val(M131)Glu creates a hydrogen bond to H-B to enhance ET to H-B. In both Phi(B) mutants, the decay kinetics of the excited primary ET donor (P*) resolve three populations with lifetimes of similar to 9 ps (50-60%), similar to 40 ps (10-20%), and similar to 200 ps (20-30%), with P+Phi(-)(B) formed predominantly from the 9 ps fraction. The 50-60% yield of P+Phi(B)- is the highest yet observed for a Phi(B)-containing RC. The results provide insight into factors needed for efficient multistep ET.

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

Science.gov (United States)

Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

2015-10-01

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. Copyright © 2015 Elsevier Inc. All rights reserved.

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

Science.gov (United States)

Crankshaw, D L; Hetnarski, K; Wilkinson, C F

1979-09-01

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species*

Science.gov (United States)

Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

2012-01-01

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b558 that donates electrons to a binuclear heme b595/heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O2, the O–O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75–0.85 per enzyme in agreement with its much higher rate of formation (∼20,000 s−1) compared with its rate of decay (∼1,900 s−1). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O–O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O–O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species. PMID:22287551

Cyanide inhibition and pyruvate-induced recovery of cytochrome c oxidase

Czech Academy of Sciences Publication Activity Database

Nůsková, Hana; Vrbacký, Marek; Drahota, Zdeněk; Houštěk, Josef

2010-01-01

Roč. 42, č. 5 (2010), s. 395-403 ISSN 0145-479X R&D Projects: GA ČR(CZ) GA303/07/0781; GA MŠk(CZ) 1M0520; GA MŠk OC08017 Institutional research plan: CEZ:AV0Z50110509 Keywords : cytochrom c oxidase * cyanide * oxygen affinity Subject RIV: CE - Biochemistry Impact factor: 3.637, year: 2010

Substrate binding in the active site of cytochrome P450cam

NARCIS (Netherlands)

Swart, M.; Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K.

2005-01-01

We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calculations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of the reaction taking place. The DFT

[ATP-synthetase activity, respiration and cytochromes of rat heart mitochondria in aging and hyperthyroidism].

Science.gov (United States)

Lemeshko, V V; Kaliman, P A; Belostotskaia, L I; Uchitel', A A

1982-04-01

The ATP-synthetase activity, the rate of oxygen uptake under different metabolic conditions, the tightness of coupling of respiration to oxidative phosphorylation and the cytochrome contents in heart mitochondria of rats from different age groups were studied under normal conditions and in hyperthyroidism. It was found that heart mitochondria of aged animals did not practically differ in terms of their functional activity from those of the young animals. Administration of thyroxin to the animals from all age groups produced no significant effects on the state of mitochondria, increasing the rate of ATP synthesis on alpha-glycerophosphate, which was especially well-pronounced in aged animals, and the cytochrome content in 1-month-old rats.

Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

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Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

2005-01-01

Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c oxidase subunit 1 mRNA; COX, cytochrome c oxidase (protein); CREB, cyclic AMP response element binding protein; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EEG, electroencephalography; EMG, electromyography; GABP, GA binding

A study on the interaction of horse heart cytochrome c with some conventional and ionic liquid surfactants probed by ultraviolet-visible and fluorescence spectroscopic techniques

Science.gov (United States)

Mondal, Satyajit; Das, Bijan

2018-06-01

The interactions of a protein cytochrome c with some selected conventional and ionic liquid surfactants have been investigated at pH 7.4 using ultraviolet-visible and fluorescence spectroscopic techniques. We used four conventional surfactants - cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), sodium N-dodecanoylsarcosinate (SDDS), and N-decanoyl-N-methylglucamine (Mega 10), and a surface active ionic liquid 1-hexadecyl-3-methylimidazolium chloride (C16MeImCl). All the investigated surfactants were found to induce an unfolding of the protein cytochrome c. In presence of CTAB, SDDS and C16MeImCl, the heme iron atom was found to loose methionine from its axial position. Differential binding of the surfactant monomers and their micelles to the protein molecules was inferred. The ionic surfactants were found to be more effective than the nonionic one in unfolding the investigated protein. However, the extent of binding of CTAB/C16MeImCl to cytochrome c reaches a plateau past the critical micellization concentration (cmc) of the surfactant. For each of the cytochrome c-DTAB, cytochrome c-SDDS and cytochrome c-Mega 10 system, although there exists an inflection in the surfactant-binding, saturation point could not be detected. It has been demonstrated from the ultraviolet-visible spectral studies that the oxidation state of iron in cytochrome c does not change when the protein binds with the investigated surfactants.

Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

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Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.; Dohnalkova, Alice; Hu, Dehong; Lemke, Rachelle A.; Piotrowski, Jeff S.; Orr, Galya; Noguera, Daniel R.; Donohue, Timothy J.

2017-05-23

Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.

IMPORTANCEThis paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase

Effect of light-dark cycles on hydrogen and poly-β-hydroxybutyrate production by a photoheterotrophic culture and Rhodobacter capsulatus using a dark fermentation effluent as substrate.

Science.gov (United States)

Montiel Corona, Virginia; Le Borgne, Sylvie; Revah, Sergio; Morales, Marcia

2017-02-01

A Rhodobacter capsulatus strain and a photoheterotrophic culture (IZT) were cultivated to produce hydrogen under different light-dark cycles. A dark fermentation effluent (DFE) was used as substrate. It was found that IZT culture had an average cumulative hydrogen production (Paccum H 2 ) of 1300±43mLH 2 L -1 under continuous illumination and light-dark cycles of 30 or 60min. In contrast, R. capsulatus reduced its Paccum H 2 by 20% under 30:30min light-dark cycles, but tripled its poly-β-hydroxybutyrate (PHB) content (308±2mgPHB gdw -1 ) compared to continuous illumination. The highest PHB content by IZT culture was 178±10mgPHB gdw -1 under 15:15min light-dark cycles. PCR-DGGE analysis revealed that the IZT culture was mainly composed of Rhodopseudomonas palustris identified with high nucleotide similarity (99%). The evaluated cultures might be used for hydrogen and PHB production. They might provide energy savings by using light-dark cycles and DFE valorization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus; the H+/H- ratio and the activation state of the enzyme during reduction of acetyl pyridine adenine dinucleotide.

Science.gov (United States)

Palmer, T; Jackson, J B

1992-02-21

Chromatophores from Rhodobacter capsulatus were incubated in the dark with NADPH and acetylpyridineadenine dinucleotide (AcPdAD+) in the presence of different concentrations of myxothiazol. The transhydrogenase activity was monitored until an appropriate mass action ratio, [AcPdAD+][NADPH]/[AcPdADH][NADP+], was reached. The sample was then illuminated and the initial rate of either AcPdAD+ reduction by NADPH or AcPdADH oxidation by NADP+ was recorded. The ratio of H+ translocated per H- equivalent transferred by transhydrogenase was calculated from the value of the membrane potential (delta pH = 0) at which illumination caused no net reaction in either direction. The mean value for the H+/H- ratio was 0.55. At greater values of [AcPdAD+][NADPH]/[AcPdADH][NADP+] than were employed in the above experiments and over a wider range of concentrations of myxothiazol, it was found that incremental increases in the membrane potential always gave rise to a decrease, never an increase in the rate of AcPdAD+ reduction. In contrast to the H(+)-ATP synthase, there is no evidence of any activation/deactivation of H(+)-transhydrogenase by the protonmotive force.

Dietary Karaya Saponin and Rhodobacter capsulatus Exert Hypocholesterolemic Effects by Suppression of Hepatic Cholesterol Synthesis and Promotion of Bile Acid Synthesis in Laying Hens

Directory of Open Access Journals (Sweden)

Sadia Afrose

2010-01-01

Full Text Available This study was conducted to elucidate the mechanism underlying the hypolipidemic action of karaya saponin or Rhodobacter (R. capsulatus. A total of 40 laying hens (20-week-old were assigned into four dietary treatment groups and fed a basal diet (as a control or basal diets supplemented with either karaya saponin, R. capsulatus, or both for 60 days. The level of serum low-density-lipoprotein cholesterol and the levels of cholesterol and triglycerides in the serum, liver, and egg yolk were reduced by all the supplementations (<.05. Liver bile acid concentration and fecal concentrations of cholesterol, triacylglycerol, and bile acid were simultaneously increased by the supplementation of karaya saponin, R. capsulatus, and the combination of karaya saponin and R. capsulatus (<.05. The supplementation of karaya saponin, R. capsulatus, and the combination of karaya saponin and R. capsulatus suppressed the incorporation of 14C from 1-14C-palmitic acid into the fractions of total lipids, phospholipids, triacylglycerol, and cholesterol in the liver in vitro (<.05. These findings suggest that the hypocholesterolemic effects of karaya saponin and R. capsulatus are caused by the suppression of the cholesterol synthesis and the promotion of cholesterol catabolism in the liver.

Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

Science.gov (United States)

Shiva, Sruti; Brookes, Paul S.; Patel, Rakesh P.; Anderson, Peter G.; Darley-Usmar, Victor M.

2001-06-01

An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

Sequential unfolding of the two-domain protein Pseudomonas stutzeri cytochrome c(4)

DEFF Research Database (Denmark)

Andersen, Niels Højmark; Jensen, Thomas Jon; Nørgaard, Allan

2002-01-01

F stutzeri cytochrome c. is a di-haem protein, composed of two globular domains each with His-Met coordinated haem. and a hydrogen bond network between the domains. The domain foldings are highly symmetric but with specific differences including structural differences of ligand coordination, and ...

Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei

NARCIS (Netherlands)

Bienen, E. J.; Maturi, R. K.; Pollakis, G.; Clarkson, A. B.

1993-01-01

The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc 1 Complex of Yeast Mitochondria

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Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc 1 complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc 1 complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc 1 complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain. PMID:21716720

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc(1) Complex of Yeast Mitochondria.

Science.gov (United States)

Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc(1) complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc(1) complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc(1) complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain.

MacA is a second cytochrome c peroxidase of Geobacter sulfurreducens.

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Seidel, Julian; Hoffmann, Maren; Ellis, Katie E; Seidel, Antonia; Spatzal, Thomas; Gerhardt, Stefan; Elliott, Sean J; Einsle, Oliver

2012-04-03

The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS(-2) as an electron donor. The observed K(M) was 38.5 ± 3.7 μM H(2)O(2) and v(max) was 0.78 ± 0.03 μmol of H(2)O(2)·min(-1)·mg(-1), resulting in a turnover number k(cat) = 0.46 · s(-1). In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.

A study on the interaction of horse heart cytochrome c with some conventional and ionic liquid surfactants probed by ultraviolet-visible and fluorescence spectroscopic techniques.

Science.gov (United States)

Mondal, Satyajit; Das, Bijan

2018-06-05

The interactions of a protein cytochrome c with some selected conventional and ionic liquid surfactants have been investigated at pH7.4 using ultraviolet-visible and fluorescence spectroscopic techniques. We used four conventional surfactants - cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), sodium N-dodecanoylsarcosinate (SDDS), and N-decanoyl-N-methylglucamine (Mega 10), and a surface active ionic liquid 1-hexadecyl-3-methylimidazolium chloride (C 16 MeImCl). All the investigated surfactants were found to induce an unfolding of the protein cytochrome c. In presence of CTAB, SDDS and C 16 MeImCl, the heme iron atom was found to loose methionine from its axial position. Differential binding of the surfactant monomers and their micelles to the protein molecules was inferred. The ionic surfactants were found to be more effective than the nonionic one in unfolding the investigated protein. However, the extent of binding of CTAB/C 16 MeImCl to cytochrome c reaches a plateau past the critical micellization concentration (cmc) of the surfactant. For each of the cytochrome c-DTAB, cytochrome c-SDDS and cytochrome c-Mega 10 system, although there exists an inflection in the surfactant-binding, saturation point could not be detected. It has been demonstrated from the ultraviolet-visible spectral studies that the oxidation state of iron in cytochrome c does not change when the protein binds with the investigated surfactants. Copyright © 2018 Elsevier B.V. All rights reserved.

Flower colour and cytochromes P450.

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Tanaka, Yoshikazu; Brugliera, Filippa

2013-02-19

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

Chemical labeling studies on isolated and vesicular bovine heart mitochondrial cytochrome c oxidase

International Nuclear Information System (INIS)

Venzke, K.S.; Reynolds, K.A.; Prochaska, L.J.

1987-01-01

Bovine heart cytochrome c oxidase dispersed in Triton X-100, Tween 80, or dodecyl maltoside was reacted with the water-soluble reagents [ 35 S]-diazonium benzene sulfonate (DABS) (10-100 μM) or [ 125 I]-iodo-DABS (34-55 nM) to map the surface topography of the enzyme in different protein aggregation states. Both reagents gave similar labeling profiles of the enzyme under all conditions. Subunits II, III, and VII were extensively labeled by DABS, while subunits I and VI were unreactive with DABS in each detergent. Subunit V exhibited an increase in DABS labeling when the enzyme was reacted in Tween 80 as compared to the enzyme in Triton X-100 or dodecyl maltoside. Also, components b and c showed an increase in DABS reactivity when the enzyme was modified in dodecyl maltoside. In general, the labeling profile of the enzyme in dodecyl maltoside resembled that of the enzyme in Triton X-100, emphasizing that the mechanism of dispersal of the enzyme by both detergents is similar. Cytochrome c oxidase incorporated into phosphatidylglycerol:phosphatidylcholine(1:20)(w:w) phospholipid vesicles (COV) by cholate dialysis was reacted with DABS and subunits II and III were significantly labeled. Approximately 65-70% of the enzyme in COV was oriented with the cytochrome c binding domain facing the extravesicular medium, as determined by comparison of the DABS labeling in subunit IV in detergent-lysed and intact COV

Atomistic simulations indicate cardiolipin to have an integral role in the structure of the cytochrome bc(1) complex

DEFF Research Database (Denmark)

Poyry, S.; Cramariuc, O.; Postila, P. A.

2013-01-01

by both ensuring the structural integrity of the protein complex and also by taking part in the proton uptake. Yet, the atom-scale understanding of these highly charged four-tail lipids in the cyt bc(1) function has remained quite unclear. We consider this issue through atomistic molecular dynamics...... the description of the role of the surrounding lipid environment: in addition to the specific CL-protein interactions, we observe the protein domains on the positive side of the membrane to settle against the lipids. Altogether, the simulations discussed in this article provide novel views into the dynamics...... simulations that are applied to the entire cyt bc(1) dimer of the purple photosynthetic bacterium Rhodobacter capsulatus embedded in a lipid bilayer. We find CLs to spontaneously diffuse to the dimer interface to the immediate vicinity of the higher potential heme b groups of the complex's catalytic Q...

The release of cytochrome c and the regulation of the programmed cell death progress in the endosperm of winter wheat (Triticum aestivum L.) under waterlogging.

Science.gov (United States)

Qi, Yuan-Hong; Mao, Fang-Fang; Zhou, Zhu-Qing; Liu, Dong-Cheng; Min-Yu; Deng, Xiang-Yi; Li, Ji-Wei; Mei, Fang-Zhu

2018-05-02

It has been shown in mammalian systems that the mitochondria can play a key role in the regulation of apoptosis by releasing intermembrane proteins (such as cytochrome c) into the cytosol. Cytochrome c released from the mitochondria to the cytoplasm activates proteolytic enzyme cascades, leading to specific nuclear DNA degradation and cell death. This pathway is considered to be one of the important regulatory mechanisms of apoptosis. Previous studies have shown that endosperm cell development in wheat undergoes specialized programmed cell death (PCD) and that waterlogging stress accelerates the PCD process; however, little is known regarding the associated molecular mechanism. In this study, changes in mitochondrial structure, the release of cytochrome c, and gene expression were studied in the endosperm cells of the wheat (Triticum aestivum L.) cultivar "huamai 8" during PCD under different waterlogging durations. The results showed that waterlogging aggravated the degradation of mitochondrial structure, increased the mitochondrial permeability transition (MPT), and decreased mitochondrial transmembrane potential (ΔΨm), resulting in the advancement of the endosperm PCD process. In situ localization and western blotting of cytochrome c indicated that with the development of the endosperm cell, cytochrome c was gradually released from the mitochondria to the cytoplasm, and waterlogging stress led to an advancement and increase in the release of cytochrome c. In addition, waterlogging stress resulted in the increased expression of the voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT), suggesting that the mitochondrial permeability transition pore (MPTP) may be involved in endosperm PCD under waterlogging stress. The MPTP inhibitor cyclosporine A effectively suppressed cell death and cytochrome c release during wheat endosperm PCD. Our results indicate that the mitochondria play important roles in the PCD of endosperm cells and that

Identification of cytochrome P450 differentiated expression related to developmental stages in bromadiolone resistance in rats (Rattus norvegicus)

DEFF Research Database (Denmark)

Markussen, Mette; Heiberg, Ann-Charlotte; Fredholm, Merete

2008-01-01

over-express the Cyp2a1 gene. TGhe altered gene expression has been suggested to be involved in the bromadiolone resistance by facilitating enhanced anticoagulant metabolism. To investigate the gene expression of these cytochrome P450 genes in rats of different developmental stages we compared...... expression profiles, from 8-, 12- and 20-week-old resistant rats of the Danish strain to profiles of anticoagulant-susceptible rats of same ages. The three age-groups were selected to represent a group of pre-pubertal, pubertal and adult rats. We found expression profiles of the pre-pubertal and pubertal...... resistant rats to concur with profiles of the adults suggesting that cytochrome P450 enzymes are involved in the Danish bromadiolone resistance regardless of developmental stage. We also investigated the relative importance of the six cytochrome P450s in the different development stages of the resistant...

4-Alkyl radical extrusion in the cytochrome P-450-catalyzed oxidation of 4-alkyl-1,4-dihydropyridines

International Nuclear Information System (INIS)

Lee, J.S.; Jacobsen, N.E.; Ortiz de Montellano, P.R.

1988-01-01

Rat liver microsomal cytochrome P-450 oxidizes the 4-methyl, 4-ethyl (DDEP), and 4-isopropyl derivatives of 3,5-bis(carbethoxy)-2,6-dimethyl-1,4,-dihydropyridine to mixtures of the corresponding 4-alkyl and 4-dealkyl pyridines. A fraction of the total microsomal enzyme is destroyed in the process. The 4-dealkyl to 4-alkyl pyridine metabolite ratio, the extent of cytochrome P-450 destruction, and the rate of spin-trapped radical accumulation are correlated in a linear inverse manner with the homolytic or heterolytic bond energies of the 4-alkyl groups of the 4-alkyl-1,4-dihydropyridines. No isotope effects are observed on the pyridine matabolite ratio, the destruction of cytochrome P-450, or the formation of ethyl radicals when [4- 2 H]DDEP is used instead of DDEP. N-Methyl- and N-ethyl-DDEP undergo N-dealkylation rather than aromatization but N-phenyl-DDEP is oxidized to a mixture of the 4-ethyl and 4-deethyl N-phenylpyridinium metabolites. In contrast to the absence of an isotope effect in the oxidation of DDEP, the 4-deethyl to 4-ethyl N-phenylpyridinium metabolite ratio increases 6-fold when N-phenyl[4- 2 H]DDEP is used. The results support the hypothesis that cytochrome P-450 catalyzes the oxidation of dihydropyridines to radical cations and show that the radical cations decay to nonradical products by multiple, substituent-dependent, mechanisms

Differential action on cancer and normal tissue by adrenochrome monoaminoguanidine methanesulfonate and cytochrome C combined with radiotherapy

International Nuclear Information System (INIS)

Nakatsugawa, S.; Sugahara, T.

1994-01-01

The possibility that radioprotective effects on potent natural killer (NK) cells by adrenochrome monoaminoguanidine methanesulfonate (AMM) + cytochrome C during radiotherapy (RT) for lung cancer might result in the radiosensitization of human lung cancer cells in vivo is examined. Human lung cancer xenografts in the right hind legs of KSN mice (10 weeks old) were locally irradiated with 20 Gy of X ray. AMM (10 mg/kg/day) and/or cytochrome C (CCC) (5 mg/kg/day) were given intraperitoneally immediately before or after RT, followed by daily administration for 4 days. Natural killer activities of host splenocytes were also tested with the standard 51 Cr releasing assay with YAC-1 cells as target cells. In a clinical study, 65 patients with lung cancer were treated with more than 50 Gy of RT with or without combination with AMM + CCC, OK-432 or AMM + CCC + OK-432. Before and after RT, lymphocyte subsets in the peripheral blood were examined with dichromatic analysis using an Ortho Spectrum IIIFCM system and fluorescent MABs. In this study, the change in the absolute number of each subset was investigated. AMM + cytochrome C augumented NK activity in KSN nude mice, protected potent NK cells in patients with lung cancer against RT and sensitized the human lung cancer xenografts to RT. AMM + cytochrome C may have potential as a differential modulator of radiosensitivity of normal tissues and of tumors. 8 refs., 2 figs., 1 tab

Benzo[alpyrene induction of cytochrome P450 1A1/1A2 in the lymph nodes of rats.

Science.gov (United States)

Borodin, Yu I; Safina, A F; Maiborodin, I V; Grishanova, A Yu

2003-12-01

Studies of mesenteric lymph nodes of rats by indirect immunoperoxidase method using monoclonal antibodies to cytochrome P450 1A/1A2 after oral dose of benzo[a]pyrene showed the presence of these cytochrome forms in monocytes, macrophages, reticular and litoral cells, cell detritus, and liquid contents of the paracortical zone and medullary substance sinuses. Oxidation of various exo- and endogenous toxins in the lymph nodes was revealed.

Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

Energy Technology Data Exchange (ETDEWEB)

Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States); Panda, Satya P., E-mail: panda@uthscsa.edu [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)

2011-08-05

Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

International Nuclear Information System (INIS)

Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue; Panda, Satya P.

2011-01-01

Highlights: → Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. → First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. → Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. → Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. → Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b 5 and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

Cytochrome P-450 complex formation in rat liver by the antibiotic tiamulin.

Science.gov (United States)

Witkamp, R F; Nijmeijer, S M; van Miert, A S

1996-01-01

Tiamulin is a semisynthetic diterpene antibiotic frequently used in farm animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds. It has been suggested that this is caused by a selective inhibition of oxidative drug metabolism via the formation of a cytochrome P-450 metabolic intermediate complex. In the present study, rats were treated orally for 6 days with tiamulin at two different doses: 40 and 226 mg/kg of body weight. For comparison, another group received 300 mg of triacetyloleandomycin (TAO) per kg, which is equivalent to the 226-mg/kg tiamulin group. Subsequently, microsomal P-450 contents, P-450 enzyme activities, metabolic intermediate complex spectra, and P-450 apoprotein concentrations were assessed. In addition, effects on individual microsomal P-450 activities were studied in control microsomes at different tiamulin and substrate concentrations. In the rats treated with tiamulin, a dose-dependent complex formation as evidenced by its absorption spectrum and an increase in cytochrome P-4503A1/2 contents as assessed by Western blotting (immunoblotting) were found. The effects were comparable to those of TAO. Tiamulin induced microsomal P-450 content, testosterone 6 beta-hydroxylation rate, erythromycin N-demethylation rate, and the ethoxyresorufin O-deethylation activity. Other activities were not affected or decreased. When tiamulin was added to microsomes of control rats, the testosterone 6 beta-hydroxylation rate and the erythromycin N-demethylation were strongly inhibited. It is concluded that tiamulin is a potent and selective inducer-inhibitor of cytochrome P-450. Though not belonging to the macrolides, the compound produces an effect on P-450 similar to those of TAO and related compounds.

Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

Science.gov (United States)

Abbrescia, Daniela Isabel; La Piana, Gianluigi; Lofrumento, Nicola Elio

2012-02-15

In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase. Copyright © 2011 Elsevier Inc. All rights reserved.

Genome-Wide Analysis, Classification, Evolution, and Expression Analysis of the Cytochrome P450 93 Family in Land Plants

OpenAIRE

Du, Hai; Ran, Feng; Dong, Hong-Li; Wen, Jing; Li, Jia-Na; Liang, Zhe

2016-01-01

Cytochrome P450 93 family (CYP93) belonging to the cytochrome P450 superfamily plays important roles in diverse plant processes. However, no previous studies have investigated the evolution and expression of the members of this family. In this study, we performed comprehensive genome-wide analysis to identify CYP93 genes in 60 green plants. In all, 214 CYP93 proteins were identified; they were specifically found in flowering plants and could be classified into ten subfamilies?CYP93A?K, with t...

Cloning, expression, crystallization and preliminary X-ray characterization of cytochrome c552 from a moderate thermophilic bacterium, Hydrogenophilus thermoluteolus

International Nuclear Information System (INIS)

Ichiki, Shin-ichi; Nakamura, Shota; Ohkubo, Tadayasu; Kobayashi, Yuji; Hasegawa, Jun; Uchiyama, Susumu; Nishihara, Hirofumi; Mizuta, Keiko; Sambongi, Yoshihiro

2005-01-01

Cytochrome c 552 of a moderate thermophile, H. thermoluteolus, was overexpressed in E. coli and crystallized for X-ray diffraction study. The amino-acid sequence of cytochrome c 552 (PH c 552 ) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogenobacter thermophilus (HT c 552 ), and from a mesophile, Pseudomonas aeruginosa (PA c 551 ). The PH c 552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coli periplasm. The overexpressed PH c 552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C222 1 , with unit-cell parameters a = 48.98, b = 57.99, c = 56.20 Å. The crystals diffract X-rays to around 2.1 Å resolution

Structure and function of the tetraheme cytochrome associated to the reaction center of Roseobacter denitrificans.

Science.gov (United States)

Garcia, D; Richaud, P; Breton, J; Verméglio, A

1994-01-01

We have characterized the tetrahemic RC bound cytochrome isolated from the quasi-photosynthetic bacterium Roseobacter denitrificans in terms of absorption spectrum, redox property and orientation with respect to the membrane plane. The heme, designated H1, which possesses the highest redox midpoint potential (+290 mV), absorbs at 555 nm. Its plane makes an angle of 40 degrees with the membrane plane. The second high potential heme, H2 (+240 mV), peaks at 554 nm and makes a tilt of 55 degrees with the membrane. The two low potential hemes, L1 and L2, present a similar and rather high redox midpoint potential (+90 mV). They absorb at 553 nm and 550 nm. One of these hemes is oriented at 40 degrees while the other makes an angle of 90 degrees with the membrane plane. The soluble cytochrome c551 completes the cyclic electron transfer between the RC and the bc1 complex. Both the oxidation and the re-reduction of cytochrome c551 are diffusible processes. Under semi-aerobic conditions, one of the low potential hemes is photo-oxidized under illumination but only extremely slowly re-reduced. This explains the requirement of high aerobic conditions for growth of Roseobacter denitrificans cells in the light.

Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

Energy Technology Data Exchange (ETDEWEB)

Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cawley, George F.; Ardoin, Taylor G. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W. [Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 (United States); Backes, Wayne L. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

2014-06-01

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

International Nuclear Information System (INIS)

Reed, James R.; Cawley, George F.; Ardoin, Taylor G.; Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W.; Backes, Wayne L.

2014-01-01

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

Characterisation of MtoD from Sideroxydans lithotrophicus: a cytochrome c electron shuttle used in lithoautotrophic growth.

Directory of Open Access Journals (Sweden)

christopher eBeckwith

2015-04-01

Full Text Available The autotrophic Sideroxydans lithotrophicus ES-1 can grow by coupling the oxidation of ferrous iron to the reduction of oxygen. Soluble ferrous iron is oxidised at the surface of the cell by an MtoAB porin-cytochrome complex that functions as an electron conduit through the outer membrane. Electrons are then transported to the cytoplasmic membrane where they are used to generate proton motive force (for ATP synthesis and NADH for autotrophic processes such as carbon fixation.As part of the mtoAB gene cluster, S. lithotrophicus also contains the gene mtoD that is proposed to encode a cytochrome c protein. We isolated mtoD from a Shewanella oneidensis expression system where the mtoD gene was expressed on a pBAD plasmid vector. Biochemical, biophysical and crystallographic characterisation of the purified MtoD revealed it as an 11 kDa monomeric protein containing a single heme. Sequence and structural alignment indicated that MtoD belonged to the class-1 cytochrome c family and had a similar fold to ferricytochrome c552 family, however the MtoD heme is bis-histidine coordinated and is substantially more exposed than the hemes of other family members. The reduction potential of the MtoD heme at pH 7 was +155 mV vs. Standard Hydrogen Electrode, which is approximately 100 mV lower than that of mitochondrial cytochromes c. Consideration of the properties of MtoD in the context of the potential respiratory partners identified from the genome suggests that MtoD could associate to multiple electron transfer partners as the primary periplasmic electron shuttle.

Electronic Connection Between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

Science.gov (United States)

Sarewicz, Marcin; Osyczka, Artur

2015-01-01

Mitochondrial respiration, an important bioenergetic process, relies on operation of four membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc1 or ubiquinol:cytochrome c oxidoreductase), provides an electronic connection between these two diffusible redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme implicate that in addition to its well-defined function of contributing to generation of proton-motive force, cytochrome bc1 may be a physiologically important point of regulation of electron flow acting as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic conditions. These features include the following: the opposing redox reactions at quinone catalytic sites located on the opposite sides of the membrane, the inter-monomer electronic connection that functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system, as well as the potential to generate superoxide and release it to the intermembrane space where it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding how cytochrome bc1 may accomplish this regulatory physiological function, what is known and remains unknown about catalytic and side reactions within the quinone binding sites and electron transfers through the cofactor chains connecting those sites with the substrate redox pools. We also discuss the developed molecular mechanisms in the context of physiology of mitochondria. PMID:25540143

Evidence that assembly of the yeast cytochrome bc1 complex involves formation of a large core structure in the inner mitochondrial membrane

Science.gov (United States)

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L.

2009-01-01

The assembly status of the cytochrome bc1 complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc1 subunits had been deleted. In all the yeast strains tested a bc1 sub-complex of about 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS-PAGE and immunodecoration, revealed the presence of the two catalytic subunits cytochrome b and cytochrome c1, associated with the non catalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Altogether these bc1 subunits build up the core structure of the cytochrome bc1 complex which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc1 core structure may represent a true assembly intermediate during the maturation of the bc1 complex, first because of its wide distribution in distinct yeast deletion strains and second for its characteristics of stability which resemble those of the intact homodimeric bc1 complex. Differently from this latter, however, the bc1 core structure is not able to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc1 complex provides a number of new elements for clarification of the molecular events leading to the maturation of the yeast cytochrome bc1 complex in the inner mitochondrial membrane. PMID:19236481

Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

Science.gov (United States)

Donnelly, Mark

2006-08-01

The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

Novel function of glutathione transferase in rat liver mitochondrial membrane: Role for cytochrome c release from mitochondria

International Nuclear Information System (INIS)

Lee, Kang Kwang; Shimoji, Manami; Hossain, Quazi Sohel; Sunakawa, Hajime; Aniya, Yoko

2008-01-01

Microsomal glutathione transferase (MGST1) is activated by oxidative stress. Although MGST1 is found in mitochondrial membranes (mtMGST1), there is no information about the oxidative activation of mtMGST1. In the present study, we aimed to determine whether mtMGST1 also undergoes activation and about its function. When rats were treated with galactosamine/lipopolysaccharide (GalN/LPS), mtMGST1 activity was significantly increased, and the increased activity was reduced by the disulfide reducing agent dithiothreitol. In mitochondria from GalN/LPS-treated rats, disulfide-linked mtMGST1 dimer and mixed protein glutathione disulfides (glutathionylation) were detected. In addition, cytochrome c release from mitochondria isolated from GalN/LPS-treated rats was observed, and the release was inhibited by anti-MGST1 antibodies. Incubation of mitochondria from control rats with diamide and diamide plus GSH in vitro resulted in dimer- and mixed disulfide bond-mediated activation of mtMGST1, respectively. The activation of mtMGST1 by diamide plus GSH caused cytochrome c release from the mitochondria, and the release was prevented by treatment with anti-MGST1 antibodies. In addition, diamide plus GSH treatment caused mitochondrial swelling accompanied by cytochrome c release, which was inhibited by cyclosporin A (CsA) and bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT) pore. Furthermore, mtMGST1 activity was also inhibited by CsA and BKA. These results indicate that mtMGST1 is activated through mixed disulfide bond formation that contributes to cytochrome c release from mitochondria through the MPT pore

Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor

DEFF Research Database (Denmark)

Jensen, Kenneth; Johnston, Jonathan B.; Montellano, Paul R. Ortiz de

2012-01-01

The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multif...

Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

Science.gov (United States)

Quester, Katrin; Juarez-Moreno, Karla; Secundino, Isamel; Roseinstein, Yvonne; Alejo, Karla P; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

2017-05-01

Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SDS-facilitated in vitro formation of a transmembrane B-type cytochrome is mediated by changes in local pH

DEFF Research Database (Denmark)

Weber, M.; Schneider, D.; Prodöhl, A.

2011-01-01

cytochrome b(559)', which can be efficiently assembled in vitro from a heme-binding PsbF homo-dimer by combining free heme with the apo-cytochrome b(559)'. Unfolding of the protein dissolved in the mild detergent dodecyl maltoside may be induced by addition of SDS, which at high concentrations leads to dimer...... dissociation. Surprisingly, absorption spectroscopy reveals that heme binding and cytochrome formation at pH 8.0 are optimal at intermediate SDS concentrations. Stopped-flow kinetics revealed that genuine conformational changes are involved in heme binding at these SDS concentrations. GPS (Global Protein...... folding State mapping) NMR measurements showed that optimal heme binding is intimately related to a change in the degree of histidine protonation. In the absence of SDS, the pH curve for heme binding is bell-shaped with an optimum at around pH 6-7. At alkaline pH values, the negative electrostatic...

Periplasmic Cytochrome c(3) of Desulfovibrio vulgaris Is Directly Involved in H2-Mediated Metal but Not Sulfate Reduction

International Nuclear Information System (INIS)

Elias, Dwayne A.; Suflita, Joseph M.; McInerney, Michael J.; Krumholz, Lee R.

2004-01-01

Kinetic parameters and the role of cytochrome c3 in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (Km 220 uM), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H2 and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H2 and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H2, lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate or pyruvate-reduced, but not H2-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H2 was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H2 was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c3 is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate

The dimerization of the yeast cytochrome bc1 complex is an early event and is independent of Rip1.

Science.gov (United States)

Conte, Annalea; Papa, Benedetta; Ferramosca, Alessandra; Zara, Vincenzo

2015-05-01

In Saccharomyces cerevisiae the mature cytochrome bc1 complex exists as an obligate homo-dimer in which each monomer consists of ten distinct protein subunits inserted into or bound to the inner mitochondrial membrane. Among them, the Rieske iron-sulfur protein (Rip1), besides its catalytic role in electron transfer, may be implicated in the bc1 complex dimerization. Indeed, Rip1 has the globular domain containing the catalytic center in one monomer while the transmembrane helix interacts with the adjacent monomer. In addition, the lack of Rip1 leads to the accumulation of an immature bc1 intermediate, only loosely associated with cytochrome c oxidase. In this study we have investigated the biogenesis of the yeast cytochrome bc1 complex using epitope tagged proteins to purify native assembly intermediates. We showed that the dimerization process is an early event during bc1 complex biogenesis and that the presence of Rip1, differently from previous proposals, is not essential for this process. We also investigated the multi-step model of bc1 assembly thereby lending further support to the existence of bona fide subcomplexes during bc1 maturation in the inner mitochondrial membrane. Finally, a new model of cytochrome bc1 complex assembly, in which distinct intermediates sequentially interact during bc1 maturation, has been proposed. Copyright © 2015 Elsevier B.V. All rights reserved.

Mapping of redox state of mitochondrial cytochromes in live cardiomyocytes using Raman microspectroscopy

DEFF Research Database (Denmark)

Brazhe, Nadezda A; Treiman, Marek; Brazhe, Alexey R

2012-01-01

This paper presents a nonivasive approach to study redox state of reduced cytochromes [Formula: see text], [Formula: see text] and [Formula: see text] of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach ...

Status of Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases.

Science.gov (United States)

Basij, M; Talebi, K; Ghadamyari, M; Hosseininaveh, V; Salami, S A

2017-02-01

Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC 50 ) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l -1 ) and acetamiprid (4.96 to 865 mg l -1 ). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR = 205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.

Betulin induces cytochrome c release and apoptosis in colon cancer cells via NOXA.

Science.gov (United States)

Zhou, Zhiyuan; Zhu, Chenfang; Cai, Zhongfang; Zhao, Feng; He, Liu; Lou, Xiaolou; Qi, Xiaoliang

2018-05-01

Betulin is a common triterpene that can be readily obtained from various plants, particularly birch trees, in their natural environment. Specific tumor cells are sensitive to betulin, whereas healthy cells are not. Betulin was observed to stimulate programmed cell death of various cancer cell lines; however, the precise molecular mechanism of action of betulin remains unknown. The present study used colon cancer cells, in which mass apoptosis triggered by betulin was identified, and the apoptotic process was demonstrated to occur via the activation of caspase-3 and -9 pathways. In addition, release of cytochrome c was detected. Furthermore, the pro-apoptotic member of the Bcl-2 protein family, NOXA, was induced following treatment with betulin, and the downregulation of NOXA markedly suppressed the release of cytochrome c and apoptosis in colon cancer cells. Conversely, the overexpression of NOXA further enhanced betulin-induced apoptosis. The present study therefore offers novel insights into the mechanism of action of the natural compound betulin against tumors.

Computer simulation and SERR detection of cytochrome c dynamics at SAM-coated electrodes

International Nuclear Information System (INIS)

Paggi, Damian Alvarez; Martin, Diego F.; Kranich, Anja; Hildebrandt, Peter; Marti, Marcelo A.; Murgida, Daniel H.

2009-01-01

In this paper we present a combined experimental and theoretical study of the heterogeneous electron transfer reaction of cytochrome c electrostatically adsorbed on metal electrodes coated with monolayers of 6-mercaptohexanoic acid. Molecular dynamics simulations and pathways calculations show that adsorption of the protein leads to a broad distribution of orientations and, thus, to a correspondingly broad distribution of electron transfer rate constants due to the orientation-dependence of the electronic coupling parameter. The adsorbed protein exhibits significant mobility and, therefore, the measured reaction rate is predicted to be a convolution of protein dynamics and tunnelling probabilities for each orientation. This prediction is confirmed by time-resolved surface enhanced resonance Raman which allows for the direct monitoring of protein (re-)orientation and electron transfer of the immobilised cytochrome c. The results provide a consistent explanation for the non-exponential distance-independence of electron transfer rates usually observed for proteins immobilized on electrodes.

Computer simulation and SERR detection of cytochrome c dynamics at SAM-coated electrodes

Energy Technology Data Exchange (ETDEWEB)

Paggi, Damian Alvarez; Martin, Diego F. [Departamento de Quimica Inorganica, Analitica y Quimica Fisica/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. 2, piso 1, C1428EHA Buenos Aires (Argentina); Kranich, Anja; Hildebrandt, Peter [Institut fuer Chemie, Technische Universitaet Berlin, Str. des 17, Juni 135, Sekr. PC14, D-10623 Berlin (Germany); Marti, Marcelo A. [Departamento de Quimica Inorganica, Analitica y Quimica Fisica/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. 2, piso 1, C1428EHA Buenos Aires (Argentina)], E-mail: marcelo@qi.fcen.uba.ar; Murgida, Daniel H. [Departamento de Quimica Inorganica, Analitica y Quimica Fisica/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. 2, piso 1, C1428EHA Buenos Aires (Argentina)], E-mail: dhmurgida@qi.fcen.uba.ar

2009-09-01

In this paper we present a combined experimental and theoretical study of the heterogeneous electron transfer reaction of cytochrome c electrostatically adsorbed on metal electrodes coated with monolayers of 6-mercaptohexanoic acid. Molecular dynamics simulations and pathways calculations show that adsorption of the protein leads to a broad distribution of orientations and, thus, to a correspondingly broad distribution of electron transfer rate constants due to the orientation-dependence of the electronic coupling parameter. The adsorbed protein exhibits significant mobility and, therefore, the measured reaction rate is predicted to be a convolution of protein dynamics and tunnelling probabilities for each orientation. This prediction is confirmed by time-resolved surface enhanced resonance Raman which allows for the direct monitoring of protein (re-)orientation and electron transfer of the immobilised cytochrome c. The results provide a consistent explanation for the non-exponential distance-independence of electron transfer rates usually observed for proteins immobilized on electrodes.

Magnitude and direction of the change in dipole moment associated with excitation of the primary electron donor in Rhodopseudomonas sphaeroides reaction centers

Energy Technology Data Exchange (ETDEWEB)

Lockhart, D.J.; Boxer, S.G.

1987-02-10

The magnitude and direction of the change in dipole moment, ..delta mu.., associated with the Q/sub y/ transition of the dimeric primary electron donor (special pair or P870) in Rhodopseudomonas sphaeroides reaction centers have been measured by Stark spectroscopy at 20 /sup 0/C. The magnitude of ..delta mu.. is found to be f/sup -1/ (10.3 +/- 0.7) D, where f is a correction factor for the local dielectric properties of the protein matrix. With the spherical cavity approximation and an effective local dielectric constant of 2, f = 1.2, and absolute value of ..delta mu.. is 8.6 +/- 0.6 D. Absolute value of ..delta mu.. for the Q/sub y/ transition of the special pair is approximately a factor of 3.4 and 2 greater than for the monomeric bacteriochlorophylls and bacteriopheophytins, respectively, in the reaction center. The angle between ..delta mu.. and the transition dipole moment for excitation of the first singlet electron state of the special pair was found to be 24 +/- 2/sup 0/. The measured values are combined to suggest a physical model in which the lowest excited singlet state of the special pair has substantial charge-transfer character and where charge is separated between the two monomers comprising the dimeric special pair. This leads to the hypothesis that the first charge-separated state in bacterial photosynthesis is formed directly upon photoexcitation. These data provide stringent values for comparison with theoretical calculations of the electronic structure of the chromophores in the reaction center.

Covalent Modification of Cytochrome C by Reactive Metabolites of Furan

OpenAIRE

Phillips, Martin B.; Sullivan, Mathilde M.; Villalta, Peter W.; Peterson, Lisa A.

2013-01-01

Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivit...

Global Analysis of Photosynthesis Transcriptional Regulatory Networks

Science.gov (United States)

Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.

2014-01-01

Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

Science.gov (United States)

Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

2015-11-20

The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol. Copyright © 2015 Elsevier B.V. All rights reserved.

Communication: Broad manifold of excitonic states in light-harvesting complex 1 promotes efficient unidirectional energy transfer in vivo

Science.gov (United States)

Sohail, Sara H.; Dahlberg, Peter D.; Allodi, Marco A.; Massey, Sara C.; Ting, Po-Chieh; Martin, Elizabeth C.; Hunter, C. Neil; Engel, Gregory S.

2017-10-01

In photosynthetic organisms, the pigment-protein complexes that comprise the light-harvesting antenna exhibit complex electronic structures and ultrafast dynamics due to the coupling among the chromophores. Here, we present absorptive two-dimensional (2D) electronic spectra from living cultures of the purple bacterium, Rhodobacter sphaeroides, acquired using gradient assisted photon echo spectroscopy. Diagonal slices through the 2D lineshape of the LH1 stimulated emission/ground state bleach feature reveal a resolvable higher energy population within the B875 manifold. The waiting time evolution of diagonal, horizontal, and vertical slices through the 2D lineshape shows a sub-100 fs intra-complex relaxation as this higher energy population red shifts. The absorption (855 nm) of this higher lying sub-population of B875 before it has red shifted optimizes spectral overlap between the LH1 B875 band and the B850 band of LH2. Access to an energetically broad distribution of excitonic states within B875 offers a mechanism for efficient energy transfer from LH2 to LH1 during photosynthesis while limiting back transfer. Two-dimensional lineshapes reveal a rapid decay in the ground-state bleach/stimulated emission of B875. This signal, identified as a decrease in the dipole strength of a strong transition in LH1 on the red side of the B875 band, is assigned to the rapid localization of an initially delocalized exciton state, a dephasing process that frustrates back transfer from LH1 to LH2.

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

Science.gov (United States)

Inui, Masayuki; Roh, Jung Hyeob; Zahn, Kenneth; Yukawa, Hideaki

2000-01-01

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria. PMID:10618203

Global analysis of photosynthesis transcriptional regulatory networks.

Directory of Open Access Journals (Sweden)

Saheed Imam

2014-12-01

Full Text Available Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888, which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

Science.gov (United States)

Beekwilder, Jules; van Houwelingen, Adèle; Cankar, Katarina; van Dijk, Aalt D J; de Jong, René M; Stoopen, Geert; Bouwmeester, Harro; Achkar, Jihane; Sonke, Theo; Bosch, Dirk

2014-02-01

Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Successful recombinant production of Allochromatium vinosum cytochrome c' requires coexpression of cmm genes in heme-rich Escherichia coli JCB712

International Nuclear Information System (INIS)

Evers, Toon H.; Merkx, Maarten

2005-01-01

Cytochrome c' from the purple photosynthetic bacterium Allochromatium vinosum (CCP) displays a unique, reversible dimer-to-monomer transition upon binding of NO, CO, and CN - . This small, four helix bundle protein represents an attractive model for the study of other heme protein biosensors, provided a recombinant expression system is available. Here we report the development of an efficient expression system for CCP that makes use of a maltose binding protein fusion strategy to enhance periplasmic expression and allow easy purification by affinity chromatography. Coexpression of cytochrome c maturase genes and the use of a heme-rich Escherichia coli strain were found to be necessary to obtain reasonable yields of cytochrome c'. Characterization using circular dichroism, UV-vis spectroscopy, and size-exclusion chromatography confirms the native-like properties of the recombinant protein, including its ligand-induced monomerization

Enzymes activities involving bacterial cytochromes incorporated in clays

International Nuclear Information System (INIS)

Lojou, E.; Giudici-Orticoni, M.Th.; Bianco, P.

2005-01-01

With the development of bio electrochemistry, researches appeared on the enzymes immobilization at the surface of electrodes for the realization of bioreactors and bio sensors. One of the main challenges is the development of host matrix able to immobilize the protein material preserving its integrity. In this framework the authors developed graphite electrodes modified by clay films. These electrodes are examined for two enzyme reactions involving proteins of sulfate-reduction bacteria. Then in the framework of the hydrogen biological production and bioreactors for the environmental pollution de-pollution, the electrochemical behavior of the cytochrome c3 in two different clays deposed at the electrode is examined

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I.

Science.gov (United States)

Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B; Erman, James E

2014-01-03

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32±0.16 M(-1) s(-1) and 0.34±0.15 s(-1), respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1±0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a "peroxygenase"-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min(-1) at pH 6.0. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular characterization of cytochrome P450 1B1 and effect of ...

African Journals Online (AJOL)

CYP1B which belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to arylhydrocarbon receptors (AhR) ligands. In this study, a new complementary DNA (cDNA) of the CYP1B subfamily ...

Evidence that the assembly of the yeast cytochrome bc1 complex involves the formation of a large core structure in the inner mitochondrial membrane.

Science.gov (United States)

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-04-01

The assembly status of the cytochrome bc(1) complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc(1) subunits were deleted. In all the yeast strains tested, a bc(1) sub-complex of approximately 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS/PAGE and immunodecoration, revealed the presence of the two catalytic subunits, cytochrome b and cytochrome c(1), associated with the noncatalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Together, these bc(1) subunits build up the core structure of the cytochrome bc(1) complex, which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc(1) core structure may represent a true assembly intermediate during the maturation of the bc(1) complex; first, because of its wide distribution in distinct yeast deletion strains and, second, for its characteristics of stability, which resemble those of the intact homodimeric bc(1) complex. By contrast, the bc(1) core structure is unable to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc(1) complex provides a number of new elements clarifying the molecular events leading to the maturation of the yeast cytochrome bc(1) complex in the inner mitochondrial membrane.

Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.).

Science.gov (United States)

Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng

2015-03-10

Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification.

Electrochemistry and biosensing activity of cytochrome c immobilized on a mesoporous interface assembled from carbon nanospheres

International Nuclear Information System (INIS)

Wang, Y.; Bian, X.; Liao, L.; Zhu, J.; Guo, K.; Kong, J.; Liu, B.

2012-01-01

We report on an amperometric biosensor for hydrogen peroxide. It is obtained via layer-by-layer assembly of ordered mesoporous carbon nanospheres and poly(diallyldimethylammonium) on the surface of an indium tin oxide (ITO) glass electrode and subsequent adsorption of cytochrome c. UV-vis absorption spectroscopy was applied to characterize the process of forming the assembled layers. Cyclic voltammetry revealed a direct and quasi-reversible electron transfer between cytochrome c and the surface of the modified ITO electrode. The surface-controlled electron transfer has an apparent heterogeneous electron-transfer rate constant (k s ) of 5.9 ± 0.2 s -1 in case of the 5-layer electrode. The biosensor displays good electrocatalytic response to the reduction of H 2O 2, and the amperometric signal increase steadily with the concentration of H 2 O 2 in the range from 5 μM to 1.5 mM. The detection limit is 1 μM at pH 7.4. The apparent Michaelis-Menten constant (K m ) of the sensor is 0.53 mM. We assume that the observation of a direct electron transfer of cytochrome c on mesoporous carbon nanospheres may form the basis for a feasible approach for durable and reliable detection of H 2 O 2 . (author)

Dietary Effect of Selenium-enriched Radish Sprouts, Vitamin E, and Rhodobacter capsulatus on Hypocholesterolemia and Immunity of Broiler

Directory of Open Access Journals (Sweden)

Tsujii H

2017-06-01

Full Text Available The study was designed to investigate the effects of dietary Selenium-enriched radish sprouts (Se-RS, Vitamin E (Vit E, and Rhodobacter capsulatus (RC on immunity, cholesterol concentration, and fatty acid composition in broiler meat. A total of 100 two-week-old male broiler chicks were randomly assigned into five dietary groups: I Control; II Se-RS (5 μg/kg Se-RS; III Se-RS+RC (5 μg/kg Se-RS + 0.2 g/kg RC; IV Se-RS+Vit E (5 μg/kg Se-RS + 50 mg/kg Vit E and V Se-RS+RC+Vit E (5 μg/kg Se-RS + 0.2 g/kg RC + 50 mg/kg Vit E. Diets and clean drinking water were offered ad libitum. After the end of 3-wk of feeding period, serum cholesterol and triglycerides concentrations were lower (P < 0.05 in broilers fed Se-RS + RC + Vit E supplemented diet compared to the control diet. At the end of the 6-wk feeding period, birds fed the Se-RS+RC+Vit E diet significantly (P < 0.05 reduced cholesterol and triglycerides concentrations and improved the ratio of unsaturated fatty acids to saturated fatty acids in broiler meat. The highest (P < 0.05 number of leukocytes was observed in broilers fed Se-RS+RC+Vit E supplemented diet. Foot web index and weights of spleen, bursa, and thymus were significantly (P < 0.05 higher in birds fed Se-RS+RC+Vit E compared to the control diet. Our findings suggest that there are dual benefits of supplementing broiler diets with Se-RS+RC+Vit E because of improvements in the bird’s immunity and meat quality that is important for health conscious consumers.

The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis.

Directory of Open Access Journals (Sweden)

Anita Loeschcke

Full Text Available Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase, LUP1 (lupeol synthase, THAS1 (thalianol synthase and MRN1 (marneral synthase derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates

Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

Science.gov (United States)

Novianty, E.; Kartikasari, L. R.; Lee, J. H.; Cahyadi, M.

2017-04-01

Meat based food products have a big opportunity to mix and adulterated with other meats. Muslim communities are prohibited to consume pork-containing product or other pig derivatives in food. Therefore, the high sensitivity, fast, cheap and accurate approach is needed to detect pig contamination in raw meat and meat-processed product such as meatball. The aim of this study was to identify pork contamination in meatball using genetic marker of mitochondrial DNA cytochrome b gene by duplex-PCR. Samples were prepared and designed by following the proportions 0, 1, 5, 10, 25% of pork in meatballs, respectively. The DNA genome was extracted from meatballs and polymerase chain reaction (PCR) was performed using species specific primer to isolate mt-DNA cytochrome b gene. The results showed that the DNA genome was successfully isolated from pork, beef, and contaminated meatballs. Furthermore, 2% agarose gels was able to visualize of duplex-PCR to identify pork contamination in meatballs up to very small proportion (1%). It can be concluded that duplex-PCR of mt-DNA cytochrome b gene was very sensitive to identify pork contamination in meatball with the presence of specific 398 bp DNA band.

Prediction of activation energies for hydrogen abstraction by cytochrome p450

DEFF Research Database (Denmark)

Olsen, Lars; Rydberg, Patrik; Rod, Thomas Holm

2006-01-01

We have estimated the activation energy for hydrogen abstraction by compound I in cytochrome P450 for a diverse set of 24 small organic substrates using state-of-the-art density functional theory (B3LYP). We then show that these results can be reproduced by computationally less demanding methods,...... of the less demanding methods are applied to study the CYP3A4 metabolism of progesterone and dextromethorphan....

In situ determination of the reduction levels of cytochromes b and c in growing bacteria : a case study with N2-fixing Azorhizobium caulinodans

NARCIS (Netherlands)

Pronk, A.F.; Boogerd, F C; Stoof, C.; Oltmann, L F; Stouthamer, A.H.; van Verseveld, H W

1993-01-01

The determination of the in situ reduction levels of cytochromes b and c in growing bacteria is achieved by coupling a chemostat with a dual wavelength spectrophotometer. Visible light absorption spectra of cytochromes present in bacterial cells actively growing in a chemostat at a specific growth

Study on the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene in humans

NARCIS (Netherlands)

Bloemen, L. J.; Monster, A. C.; Kezic, S.; Commandeur, J. N.; Veulemans, H.; Vermeulen, N. P.; Wilmer, J. W.

2001-01-01

To investigate in humans the contribution of the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene (TRI) under controlled repeated exposure in volunteers, and under occupational conditions. Volunteers were exposed to TRI, using repeated 15 min exposures at 50 and 100

Expression of cytochrome P450 genes in CD34(+) hematopoietic stem and progenitor cells

Czech Academy of Sciences Publication Activity Database

Souček, P.; Anzenbacher, P.; Skoumalová, I.; Dvořák, Michal

2005-01-01

Roč. 23, č. 9 (2005), s. 1417-1422 ISSN 1066-5099 Institutional research plan: CEZ:AV0Z50520514 Keywords : CD34+ stem/progenitor cells * cytochrome P450 isoforms Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.094, year: 2005