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R. Haser - One of the best experts on this subject based on the ideXlab platform.
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Key Role of Phenylalanine 20 in Cytochrome C3: Structure, Stability, and Function Studies†
Biochemistry, 1999Co-Authors: Alain Dolla, R. Haser, Mirjam Czjzek, P. Arnoux, I. Protasevich, V. Lobachov, Marianne Brugna, ‡ Marie Thérèse Giudici-orticoni, And Alexander A. Makarov, M. BruschiAbstract:Aromatic residues in c-type Cytochromes might have an important function in the folding and/or electron transferring properties of the molecule. In the tetraheme Cytochrome C3 (Mr 13 000) from Desulfovibrio vulgaris Hildenborough, Phe20, is located between heme 1 and heme 3 with its aromatic ring close and almost parallel to the ring plane of heme 1. We replaced this residue by a nonaromatic hydrophobe residue, leucine, and analyzed the effects in terms of functional, structural, and physicochemical properties. While the F20L replacement did not have any strong effects on the heme region stability, a decrease of the thermostability of the whole molecule was observed. In the same way, the four macroscopic redox potentials were affected by the mutation as well as the flexibility of the surface loop around heme 4. The F20L replacement itself and/or this structural modification might be responsible for the loss of the intermolecular cooperativity between F20L Cytochrome C3 molecules.
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Key role of phenylalanine 20 in Cytochrome C3: structure, stability, and function studies.
Biochemistry, 1999Co-Authors: A Dolla, R. Haser, Mirjam Czjzek, Mt. Giudici-orticoni, P. Arnoux, I. Protasevich, V. Lobachov, M. Brugna, A. Makarov, M. BruschiAbstract:Aromatic residues in c-type Cytochromes might have an important function in the folding and/or electron transferring properties of the molecule. In the tetraheme Cytochrome C3 (Mr 13 000) from Desulfovibrio vulgaris Hildenborough, Phe20, is located between heme 1 and heme 3 with its aromatic ring close and almost parallel to the ring plane of heme 1. We replaced this residue by a nonaromatic hydrophobe residue, leucine, and analyzed the effects in terms of functional, structural, and physicochemical properties. While the F20L replacement did not have any strong effects on the heme region stability, a decrease of the thermostability of the whole molecule was observed. In the same way, the four macroscopic redox potentials were affected by the mutation as well as the flexibility of the surface loop around heme 4. The F20L replacement itself and/or this structural modification might be responsible for the loss of the intermolecular cooperativity between F20L Cytochrome C3 molecules.Aromatic residues in c-type Cytochromes might have an important function in the folding and/or electron transferring properties of the molecule. In the tetraheme Cytochrome C3 (Mr 13 000) from Desulfovibrio vulgaris Hildenborough, Phe20, is located between heme 1 and heme 3 with its aromatic ring close and almost parallel to the ring plane of heme 1. We replaced this residue by a nonaromatic hydrophobe residue, leucine, and analyzed the effects in terms of functional, structural, and physicochemical properties. While the F20L replacement did not have any strong effects on the heme region stability, a decrease of the thermostability of the whole molecule was observed. In the same way, the four macroscopic redox potentials were affected by the mutation as well as the flexibility of the surface loop around heme 4. The F20L replacement itself and/or this structural modification might be responsible for the loss of the intermolecular cooperativity between F20L Cytochrome C3 molecules.
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Interfacial properties of the polyheme Cytochrome C3 superfamily from Desulfovibrio.
Biochemistry, 1995Co-Authors: L. Florens, R. Haser, Mirjam Czjzek, A Dolla, M. Ivanova, R. Verger, M. BruschiAbstract:In order to compare the interfacial behavior of the polyheme Cytochromes c which belong to the Cytochrome C3 superfamily, the monomolecular film technique was used to determine whether and how these metalloproteins interact with (phospho)lipids). Measurements of the variations of surface pressure and surface potential versus time have shown differences in their penetration capacity into phosphatidylcholine, dicaprin, and phosphatidylglycerol films. The Desulfovibrio vulgaris Hildenborough Cytochrome with 16 hemes (Hmc) and Desulfovibrio desulfuricans Norway tetra- and octaheme Cytochromes C3, which have been assumed to be soluble periplasmic molecules, may be considered as extrinsic membrane proteins, unlike the D. vulgaris Hildenborough Cytochrome C3 (Mr 13 000). The interfacial properties are discussed in terms of the available three-dimensional structural data, the electrostatic potential calculation, and the results obtained by hydrophobic cluster analysis of the Cytochrome sequences. The very different behavior of the two Cytochromes C3 (Mr 13 000) enlightens the role of a particular surface loop in the interaction with a model membrane. A functional interpretation is proposed assuming that the D. vulgaris Hildenborough Hmc and both Cytochromes C3 (Mr 13 000) and (Mr 26 000) from the Norway strain might provide the link between periplasmic hydrogen oxidation and cytoplasmic sulfate reduction.In order to compare the interfacial behavior of the polyheme Cytochromes c which belong to the Cytochrome C3 superfamily, the monomolecular film technique was used to determine whether and how these metalloproteins interact with (phospho)lipids). Measurements of the variations of surface pressure and surface potential versus time have shown differences in their penetration capacity into phosphatidylcholine, dicaprin, and phosphatidylglycerol films. The Desulfovibrio vulgaris Hildenborough Cytochrome with 16 hemes (Hmc) and Desulfovibrio desulfuricans Norway tetra- and octaheme Cytochromes C3, which have been assumed to be soluble periplasmic molecules, may be considered as extrinsic membrane proteins, unlike the D. vulgaris Hildenborough Cytochrome C3 (Mr 13 000). The interfacial properties are discussed in terms of the available three-dimensional structural data, the electrostatic potential calculation, and the results obtained by hydrophobic cluster analysis of the Cytochrome sequences. The very different behavior of the two Cytochromes C3 (Mr 13 000) enlightens the role of a particular surface loop in the interaction with a model membrane. A functional interpretation is proposed assuming that the D. vulgaris Hildenborough Hmc and both Cytochromes C3 (Mr 13 000) and (Mr 26 000) from the Norway strain might provide the link between periplasmic hydrogen oxidation and cytoplasmic sulfate reduction.
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Crystal structure of Cytochrome C3 from Desulfovibrio desulfuricans Norway at 1.7 A resolution.
Journal of molecular biology, 1994Co-Authors: Mirjam Czjzek, Mireille Bruschi, Françoise Guerlesquin, Françoise Payan, R. HaserAbstract:Abstract The crystal structure of Cytochrome C3 (Mr 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0·198 covering the range from 5·5 to 1·7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometery and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended α-helix, a short β-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the Cytochrome C3 from Desulfovibrio vulgaris Miyazaki.
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Crystal structure of Cytochrome C3 from Desulfovibrio desulfuricans Norway at 1.7 A resolution.
Journal of Molecular Biology, 1994Co-Authors: Mirjam Czjzek, M. Bruschi, F. Payan, F. Guerlesquin, R. HaserAbstract:The crystal structure of Cytochrome C3 (M(r) 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a simulated annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0.198 covering the range from 5.5 to 1.7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometry and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended alpha-helix, a short beta-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the Cytochrome C3 from Desulfovibrio vulgaris Miyazaki.The crystal structure of Cytochrome C3 (M(r) 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a simulated annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0.198 covering the range from 5.5 to 1.7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometry and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended alpha-helix, a short beta-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the Cytochrome C3 from Desulfovibrio vulgaris Miyazaki.
Hideo Akutsu - One of the best experts on this subject based on the ideXlab platform.
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Specific binding of CO to tetraheme Cytochrome C3.
Biochemistry, 2006Co-Authors: Yuki Takayama, Naoki Yahata, Takashi Saitoh, Yukiko Kobayashi, Hiroshi Hori, Takahisa Ikegami, Hideo AkutsuAbstract:Carbon monoxide (CO) has been identified as another bioactive molecule like NO. Binding of CO to a tetraheme Cytochrome C3 (cyt C3) was investigated using visible absorption spectroscopy, circular dichroism (CD), and NMR. CO was found to bind to the four hemes in different manners. CD spectra, however, indicated that only single-site CO binding can keep the protein intact. The Kd for the single-site binding was 8.0 μM, which is a typical value for a CO sensor protein. Furthermore, NMR spectra of uniformly 15N-labeled and specifically [15N]His-labeled proteins have provided evidence that CO specifically binds to the sixth coordination site of heme 2 via single-site binding. The CO-bound cyt C3 could conduct redox reactions. In light of triheme Cytochrome c7, the CO-bound cyt C3 may work as an electron transporter. It was reported for sulfate-reducing bacteria that CO can be used as an energy source and CO cycling is operating like H2 cycling. Therefore, the CO-bound cyt C3 may play a role in maintaining el...
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Redox interaction of Cytochrome C3 with [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.
Biochemistry, 2006Co-Authors: Naoki Yahata, Takashi Saitoh, Yuki Takayama, Kiyoshi Ozawa, Hideaki Ogata, Yoshiki Higuchi, Hideo AkutsuAbstract:Cytochrome C3 isolated from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F, is a tetraheme protein. Its physiological partner, [NiFe] hydrogenase, catalyzes the reversible oxidoreduction of molecular hydrogen. To elucidate the mechanism of electron transfer between Cytochrome C3 and [NiFe] hydrogenase, the transient complex formation by these proteins was investigated by means of NMR. All NH signals of uniformly 15N-labeled ferric Cytochrome C3 except N-terminus, Pro, and Gly73 were assigned. 1H-15N HSQC spectra were recorded for 15N-labeled ferric and ferrous Cytochrome C3, in the absence and presence of hydrogenase. Chemical shift perturbations were observed in the region around heme 4 in both oxidation states. Additionally, the region between hemes 1 and 3 in ferrous Cytochrome C3 was affected in the presence of hydrogenase, suggesting that the mode of interaction is different in each redox state. Heme 3 is probably the electron gate for ferrous Cytochrome C3. To investigate the transi...
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roles of noncoordinated aromatic residues in redox regulation of Cytochrome C3 from desulfovibrio vulgaris miyazaki f
Biochemistry, 2004Co-Authors: Yuki Takayama, Kiyoshi Ozawa, Erisa Harada, Rie Kobayashi, Hideo AkutsuAbstract:The roles of aromatic residues in redox regulation of Cytochrome C3 were investigated by site-directed mutagenesis at every aromatic residue except for axial ligands (Phe20, Tyr43, Tyr65, Tyr66, Hi...
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Role of the Aromatic Ring of Tyr43 in Tetraheme Cytochrome C3 from Desulfovibrio vulgaris Miyazaki F
Biophysical journal, 2003Co-Authors: Kiyoshi Ozawa, Yuki Takayama, Hideaki Ogata, Yoshiki Higuchi, Michael A. Cusanovich, Fumiko Yasukawa, Tomoaki Ohmura, Yusuke Tomimoto, Hideo AkutsuAbstract:Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme Cytochrome C3. The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of Cytochrome C3. Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L Cytochrome C3 by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.
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Ionic Strength-Dependent Physicochemical Factors in Cytochrome C3 Regulating the Electron Transfer Rate
Biophysical journal, 1998Co-Authors: Tomoaki Ohmura, Michael A. Cusanovich, Katsumi Niki, Haruki Nakamura, Hideo AkutsuAbstract:The effect of ionic strength on the macroscopic and microscopic redox potentials and the heme environment of Cytochrome C3 from Desulfovibrio vulgaris Miyazaki F have been investigated by NMR and electrochemical methods. The redox potentials of this tetraheme protein are found to be ionic strength-dependent. Especially, the microscopic redox potentials of hemes 2 and 3 at the fourth reduction step increase significantly with increasing ionic strength, which is in contraction to the theoretical expectation. The coordinated imidazole proton signals are unaffected by ionic strength. However, the methyl and propionate proton signals of hemes 1 and 4 showed significant ionic strength dependencies that are distinct from those for hemes 2 and 3. This heme classification is the same as that found in the ionic strength dependencies of the microscopic redox potentials at the fourth reduction step. Furthermore, the effect of ionic strength on the electrostatic potentials at the heme irons has been examined on the theoretical basis. The electrostatic potential at heme 4 changes up to 1 M ionic strength, which was not expected from the observations reported on Cytochromes so far. These results are discussed in connection with the reported anomalous ionic strength dependency of the reduction rate of Cytochrome C3.
Mireille Bruschi - One of the best experts on this subject based on the ideXlab platform.
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Interaction and electron transfer between the high molecular weight Cytochrome and Cytochrome C3 from Desulfovibrio vulgaris Hildenborough: kinetic, microcalorimetric, EPR and electrochemical studies.
Biochimica et biophysica acta, 2005Co-Authors: Marianne Guiral, Mireille Bruschi, Pierre Bianco, Bruno Guigliarelli, Gisèle Leroy, Philippe Gallice, Wolfgang Nitschke, Marie-thérèse Giudici-orticoniAbstract:The complex formation between the tetraheme Cytochrome C3 and hexadecaheme high molecular weight Cytochrome c (Hmc), the structure of which has recently been resolved, has been characterized by cross-linking experiments, EPR, electrochemistry and kinetic analysis, and some key parameters of the interaction were determined. The analysis of electron transfer between [Fe] hydrogenase, Cytochrome C3 and Hmc demonstrates a redox-shuttling role of Cytochrome C3 in the pathway from hydrogenase to Hmc, and shows an effect of redox state on the interaction between the two Cytochromes. The role of polyheme Cytochromes in electron transfer from periplasmic hydrogenase to membrane redox proteins is assessed. A model with Cytochrome C3 as an intermediate between hydrogenase and various polyheme Cytochromes is proposed and its physiological consequences are discussed.
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Amperometric Cytochrome C3-based biosensor for chromate determination.
Biosensors and Bioelectronics, 2003Co-Authors: C. Michel, Canh Tran Minh, Mireille Bruschi, Fabienne Battaglia-brunet, Ioannis IgnatiadisAbstract:The chromate reductase activity of Cytochrome C3 (Cyt C3, Mr 13 000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO42−) bioavailability. The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied. Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode). Immobilization of Cyt C3 by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited. The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l−1 (3.85–132 μM) with a sensitivity of 35 nA mg−1 l (1.82 nA μM−1). No interference was observed with As(V), As(III) and Fe(III). Only a small amount of Cyt C3 (372 ng of protein) was needed for this biosensor.
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Kinetics and interaction studies between Cytochrome C3 and Fe-only hydrogenase from Desulfovibrio vulgaris hildenborough
Proteins, 1998Co-Authors: Marianne Brugna, Marie-thérèse Giudici-orticoni, Silvia Spinelli, Kieron Brown, Mariella Tegoni, Mireille BruschiAbstract:Hydrogenases from Desulfovibrio are found to catalyze hydrogen uptake with low potential multiheme Cytochromes, such as Cytochrome C3, acting as acceptors. The production of Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough was improved with respect to the growth phase and media to determine the best large-scale bacteria growth conditions. The interaction and electron transfer from Fe-only hydrogenase to multiheme Cytochrome has been studied in detail by both BIAcore and steady-state measurements. The electron transfer between [Fe] hydrogenase and Cytochrome C3 appears to be a cooperative phenomenon (h = 1.37). This behavior could be related to the conductivity properties of multihemic Cytochromes. An apparent dissociation constant was determined (2 × 10-7 M). The importance of the cooperativity for contrasting models proposed to describe the functional role of the hydrogenase/Cytochrome C3 complex is discussed. Presently, the only determined structure is from [NiFe] hydrogenase and there are no obvious similarities between [NiFe] and [Fe] hydrogenase. Furthermore, no crystallographic data are available concerning [Fe] hydrogenase. The first results on crystallization and X-ray crystallography are reported. Proteins 33:590–600, 1998. © 1998 Wiley-Liss, Inc.
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A single mutation in the heme 4 environment of Desulfovibrio desulfuricans Norway Cytochrome C3 (Mr 26,000) greatly affects the molecule reactivity.
The Journal of biological chemistry, 1997Co-Authors: Corinne Aubert, Judy D. Wall, Mireille Bruschi, Gisèle Leroy, Alain DollaAbstract:The gene encoding Desulfovibrio desulfuricans Norway Cytochrome C3 (Mr 26,000), a dimeric octaheme Cytochrome belonging to the polyheme Cytochrome C3 superfamily, has been cloned and successfully expressed in another sulfate reducing bacteria, D. desulfuricans G201. The gene, named cycD, is monocistronic and encodes a Cytochrome precursor of 135 amino acids with an extension at the NH2 terminus of 24 amino acids. This extension acts as a signal sequence which allows export across the cytoplasmic membrane into the periplasmic space. Tyrosine 73, which is in a close contact with the histidine sixth axial ligand to the heme 4 iron atom, has been replaced by a glutamate residue using site-directed mutagenesis. The Cytochrome mutant when expressed in D. desulfuricans G201, is correctly folded and matured. A global increase of the oxidoreduction potentials of about 50 mV is measured for the Y73E Cytochrome. The mutation also has a strong influence on the interaction of the Cytochrome with its redox partner, the hydrogenase. This suggests, like the tetraheme Cytochrome C3 (Mr 13, 000), heme 4 is the interactive heme in the Cytochrome-hydrogenase complex and that alteration of the heme 4 environment can greatly affect the electron transfer reaction with its redox partner.
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Crystal structure of Cytochrome C3 from Desulfovibrio desulfuricans Norway at 1.7 A resolution.
Journal of molecular biology, 1994Co-Authors: Mirjam Czjzek, Mireille Bruschi, Françoise Guerlesquin, Françoise Payan, R. HaserAbstract:Abstract The crystal structure of Cytochrome C3 (Mr 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0·198 covering the range from 5·5 to 1·7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometery and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended α-helix, a short β-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the Cytochrome C3 from Desulfovibrio vulgaris Miyazaki.
Françoise Guerlesquin - One of the best experts on this subject based on the ideXlab platform.
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The Cytochrome C3-[Fe]-hydrogenase electron-transfer complex: structural model by NMR restrained docking.
FEBS Letters, 2003Co-Authors: Latifa Elantak, Olivier Bornet, Xavier Morelli, Alain Dolla, Mirjam Czjzek, Claude E. Hatchikian, Françoise GuerlesquinAbstract:Abstract Cytochrome C3 (Mr 13 000) is a low redox potential Cytochrome specific of the anaerobic metabolism in sulfate-reducing bacteria. This tetrahemic Cytochrome is an intermediate between the [Fe]-hydrogenase and the Cytochrome Hmc in Desulfovibrio vulgaris Hildenborough strain. The present work describes the structural model of the Cytochrome C3–[Fe]-hydrogenase complex obtained by nuclear magnetic resonance restrained docking. This model connects the distal cluster of the [Fe]-hydrogenase to heme 4 of the Cytochrome, the same heme found in the interaction with Cytochrome Hmc. This result gives evidence that Cytochrome C3 is an electron shuttle between the periplasmic hydrogenase and the Hmc membrane-bound complex.
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Letter to the Editor: Sequential NMR assignment of the ferri-Cytochrome C3 from Desulfovibrio vulgaris Hildenborough
Journal of Biomolecular NMR, 2002Co-Authors: Latifa Elantak, Olivier Bornet, Xavier Morelli, Alain Dolla, Françoise GuerlesquinAbstract:Cytochromes C3 are low redox potential Cytochromes involved in anaerobic metabolism. These periplasmic proteins contain four bi-histidinyl coordinated hemes. Multiheme Cytochromes have been found in sulfate reducing bacteria of the genus Desulfovibrio. Preliminary analysis of Desulfovibrio vulgaris Hildenborough genome pointed out the existence of several putative genes encoding tetraheme Cytochromes (http://www.tigr.org). Analysis of their genomic context showed that some of them are isolated (i.e., the gene encoding the well known soluble Cytochrome C3 (Mr 13,000)) while others are part of multienzymatic complexes (i.e., the tetraheme Cytochrome subunit in formate dehydrogenase (Sebban et al., 1995)). The large diversity of the tetraheme Cytochromes in this organism must be correlated with the great specificity of these molecules for their oxidoreduction partners. We have recently reported a new approach to study electron transfer complexes combining NMR spectroscopy and theoretical calculations. 1H-15N HSQC are performed on an 15N-labelled redox partner, and we use the chemical shift variations induced upon complex formation to map the interacting site and to filter the ab initio models obtained by Bigger (Morelli et al., 2000). 1H-15N HSQC assignment is thus the first step of the functional study of Cytochromes C3. We have initiated our studies with the soluble Cytochrome C3 (Mr 13,000). The gene of this protein was cloned and sequenced (Voordouw et al., 1985) and the structure of this tetraheme Cytochrome was solved by x-ray (Matias et al., 1993).
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Crystal structure of Cytochrome C3 from Desulfovibrio desulfuricans Norway at 1.7 A resolution.
Journal of molecular biology, 1994Co-Authors: Mirjam Czjzek, Mireille Bruschi, Françoise Guerlesquin, Françoise Payan, R. HaserAbstract:Abstract The crystal structure of Cytochrome C3 (Mr 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0·198 covering the range from 5·5 to 1·7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometery and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended α-helix, a short β-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the Cytochrome C3 from Desulfovibrio vulgaris Miyazaki.
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Characterization and oxidoreduction properties of Cytochrome C3 after heme axial ligand replacements.
The Journal of biological chemistry, 1994Co-Authors: Alain Dolla, Pierre Bianco, Jean Haladjian, Françoise Guerlesquin, Jd Wall, Laurence Florens, G Voordouw, Eric Forest, Mireille BruschiAbstract:Abstract Cytochrome C3 (M(r) 13,000) is a tetrahemic Cytochrome in which the four heme iron atoms are coordinated by 2 histidine residues at the axial positions. The presence of several oxidoreduction centers in the same molecule raises the question of their coupling. To investigate this mechanism, four single mutations were introduced in Cytochrome C3 by site-directed mutagenesis, leading to the replacement of each histidine, the sixth axial ligand of the heme iron atom, by a methionine residue. Characterization of the new set of molecules using biochemical and biophysical techniques was carried out. The novel methionine was correctly coordinated to the iron atom of hemes 3 and 4 in H25M and H70M Cytochromes C3, respectively, and this coordination induced a large increase in the oxidoreduction potential of the mutated heme. In contrast, in the case of H22M and H35M Cytochromes C3, in which the corresponding methionine is in an oxidized form, only slight changes in redox potential values were observed. In H22M, H25M, and H35M Cytochromes C3, two conformations of the molecule were possible, in which the methionine is either free or coordinated to the iron atom. The rate constants for the electron exchange reactions between the Cytochrome mutants and the hydrogenase were measured using electrochemical techniques. Distinct behaviors were revealed depending on the mutation. The values of the rate constants for the electron exchange reactions are interpreted in terms of intramolecular electron exchange among the four hemes of the Cytochrome.
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Reactivity of [Fe] and [NiFeSe] hydrogenases with their oxido-reduction partner: The tetraheme Cytochrome C3
Biochemical and biophysical research communications, 1992Co-Authors: Pierre Bianco, Mireille Bruschi, Jean Haladjian, Françoise GuerlesquinAbstract:In order to understand the electron transfer mechanisms for the [Fe] and [NiFe] hydrogenases, a kinetic study of Cytochrome C3 reduction has been undertaken. Cyclic voltammetry and controlled-potential amperometry techniques have been used to investigate the intermolecular electron-transfer reaction between Cytochrome C3 and [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough. Electron-transfer cross-reactions between [Fe] or [NiFeSe] hydrogenase and Cytochrome C3 from Desulfovibrio vulgaris Hildenborough or Desulfovibrio desulfuricans Norway have been studied. Some structural implications are considered from these experimental data.
Jean Legall - One of the best experts on this subject based on the ideXlab platform.
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a membrane bound Cytochrome C3 a type ii Cytochrome C3 from desulfovibrio vulgaris hildenborough
ChemBioChem, 2001Co-Authors: Filipa M A Valente, Ligia M Saraiva, Antonio V Xavier, Jean Legall, Miguel Teixeira, Ines A C PereiraAbstract:A new tetraheme Cytochrome C3 was isolated from the membranes of Desulfovibrio vulgaris Hildenborough (DvH). This Cytochrome has a molecular mass of 13.4 kDa and a pI of 5.5 and contains four heme c groups with apparent reduction potentials of -170 mV, -235 mV, -260 mV and -325 mV at pH 7.6. The complete sequence of the new Cytochrome, retrieved from the preliminary data of the DvH genome, shows that this Cytochrome is homologous to the "acidic" Cytochrome C3 from Desulfovibrio africanus (Da). A model for the structure of the DvH Cytochrome was built based on the structure of the Da Cytochrome. Both Cytochromes share structural features that distinguish them from other Cytochrome C3 proteins, such as a solvent-exposed heme 1 surrounded by an acidic surface area, and a heme 4 which lacks most of the surface lysine patch proposed to be the site of hydrogenase interaction in other Cytochrome C3 proteins. Furthermore, in contrast to previously discovered Cytochrome C3 proteins, the genes coding for these two Cytochromes are adjacent to genes coding for two membrane-associated FeS proteins, which indicates that they may be part of membrane-bound oxidoreductase complexes. Altogether these observations suggest that the DvH and Da Cytochromes are a new type of Cytochrome C3 proteins (Type II: TpII-C3) with different redox partners and physiological function than the other Cytochrome C3 proteins (Type I: TpI-C3). The DvH TpII-C3 is reduced at considerable rates by the two membrane-bound [NiFe] and [NiFeSe] hydrogenases, but catalytic amounts of TpI-C3 increase these rates two- and fourfold, respectively. With the periplasmic [Fe] hydrogenase TpII-C3 is reduced much slower than TpI-C3, and no catalytic effect of TpI-C3 is observed.
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Reevaluation of the redox and redox-Bohr cooperativity in tetrahaem Desulfovibrio vulgaris (Miyazaki F) Cytochrome C3
JBIC Journal of Biological Inorganic Chemistry, 1997Co-Authors: Carlos A. Salgueiro, Antonio V Xavier, Jean Le Gall, David L. Turner, Jean LegallAbstract:The thermodynamic model of five interacting charge centres (four haems and an ionisable centre), which was used in the characterisation of the thermodynamic properties of Desulfovibrio vulgaris (Hildenborough) Cytochrome C3 (C3DvH), is now used to reevaluate the thermodynamic properties in Desulfovibrio vulgaris (Miyazaki F) Cytochrome C3 (C3DvM) on the basis of published data (Park, J.-S., Ohmura, T., Kano, K., Sagara, T., Niki, K., Kyogoku, Y. and Akutsu, H. (1996) Biochim. Biophys. Acta 1293, 45–54). Contrary to the assertion of Park et al. (1996), the pH dependence of the proton chemical shifts of haem methyls in C3DvM in several stages of oxidation is well described by the model, which involves both homotropic (e–/e–) and heterotropic (e–/H+) cooperativity. This shows that the pH dependence observed for C3DvM is not significantly more complicated than that observed for C3DvH. Since the parameters which we now obtain for C3DvM are generated with the same model as those from C3DvH, albeit using less precise data, it is possible to make a preliminary comparison of the thermodynamic properties of these two proteins and of their role in energy transduction.
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nmr studies of cooperativity in the tetrahaem Cytochrome C3 from desulfovibrio vulgaris
FEBS Journal, 1996Co-Authors: Teresa Catarino, Jean Legall, Carlos A. Salgueiro, David L. Turner, Antonio V XavierAbstract:The thermodynamic properties of the Desulfovibrio vulgaris (Hildenborough) tetrahaem Cytochrome C3 (DvC3) are rationalised by a model which involves both homotropic (e−/e−) and heterotropic (e−/H+) cooperativity. The paramagnetic shifts of a methyl group from each haem of the DVC3 have been determined in each stage of oxidation at several pH values by means of two-dimensional exchange NMR. The thermodynamic parameters are obtained by fitting the model to the NMR data and to redox titrations followed by visible spectroscopy. They show significant positive cooperativity between two of the haems whereas the remaining interactions appear to be largely electrostatic in origin. These parameters imply that the protein undergoes a proton-assisted two-electron transfer which can be used for energy transduction. Comparison with the crystal structure together with measurement of the kinetics of proton exchange suggest that the pH dependence is mediated by a charged residue(s) readily acessible to the solvent and close to haem I.
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Structural and functional characterization of Cytochrome C3 from D. desulfuricans ATCC 27774 by 1H‐NMR
FEBS letters, 1996Co-Authors: Ricardo O. Louro, Jean Legall, David L. Turner, Isabel Pacheco, Antonio V XavierAbstract:Abstract Cooperativity between redox and protonation centres is known to be crucial for the function of complex proteins, but it is often difficult to describe in terms of thermodynamic parameters. Cytochrome C3 is a good model for these studies since, while retaining the overall complexity of larger systems, it is suitable for detailed crystallographic and spectroscopic studies. Assignment of the haem substituent NMR resonances, together with NMR redox titrations of Cytochrome C3 from D. desulfuricans ATCC 27774, was used to correlate relative redox potentials to specific haems in the structure: haem II ≊ haem I haem IV haem III . This order is different from that determined for the homologous proteins studied and in disagreement with that previously reported for this Cytochrome (Morais, J., Palma, N., Frazao, C., Caldeira, J., LeGall, J., Moura, I., Moura, J.J.G. and Carrondo, M.A. (1995) Biochemistry 34, 12830–12841).
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Redox-Bohr effect in the tetrahaem Cytochrome C3 from Desulfovibrio vulgaris: a model for energy transduction mechanisms
JBIC Journal of Biological Inorganic Chemistry, 1996Co-Authors: Ricardo O. Louro, Teresa Catarino, Jean Legall, Carlos A. Salgueiro, Antonio V XavierAbstract:Using potentiometric titrations, two protons were found to participate in the redox-Bohr effect observed for Cytochrome C3 from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence of a concerted proton-assisted 2e– step, ideally suited for the coupling role of Cytochrome C3 to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H2 as energy source, Cytochrome C3 can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of Cytochrome c oxidase.
