Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 32
Filter
Add more filters










Publication year range
1.
Extremophiles ; 25(4): 385-392, 2021 Jul.
Article in English | MEDLINE | ID: mdl-34196828

ABSTRACT

In piezophilic microorganisms, enzymes are optimized to perform under high hydrostatic pressure. The two major reported mechanisms responsible for such adaptation in bacterial species are changes in amino acids in the protein structure, favoring their activity and stability under high-pressure conditions, and the possible accumulation of micromolecular co-solutes in the cytoplasm. Recently, the accumulation of glutamate in the cytoplasm of piezophilic Desulfovibrio species has been reported under high-pressure growth conditions. In this study, analysis of the effect of glutamate on the enzymatic activity of the thioredoxin reductase/thioredoxin enzymatic complex of either a piezosensitive or a piezophilic microorganism confirms its role as a protective co-solute. Analysis of the thioredoxin structures suggests an adaptation both to the presence of glutamate and to high hydrostatic pressure in the enzyme from the piezophilic strain. Indeed, the presence of large surface pockets could counterbalance the overall compression that occurs at high hydrostatic pressure to maintain enzymatic activity. A lower isoelectric point and a greater dipolar moment than that of thioredoxin from the piezosensitive strain would allow the protein from the piezophilic strain to compensate for the presence of the charged amino acid glutamate to interact with its partner.


Subject(s)
Desulfovibrio , Glutamic Acid , Adaptation, Physiological , Hydrostatic Pressure , Thioredoxins
2.
Microbiology (Reading) ; 159(Pt 12): 2663-2673, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24085836

ABSTRACT

Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.


Subject(s)
Desulfovibrio vulgaris/metabolism , Hydrogen/metabolism , Membrane Proteins/metabolism , Oxidoreductases/metabolism , Oxygen/metabolism , Periplasm/metabolism , Sulfates/metabolism , Anaerobiosis , Desulfovibrio vulgaris/enzymology , Electron Transport , Gene Deletion , Membrane Proteins/genetics , Microbial Viability , Oxidation-Reduction , Oxidoreductases/genetics , Periplasm/enzymology
3.
Microbiology (Reading) ; 157(Pt 9): 2720-2732, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21737501

ABSTRACT

Although Desulfovibrio vulgaris Hildenborough (DvH) is a strictly anaerobic bacterium, it is able to consume oxygen in different cellular compartments, including extensive periplasmic O2 reduction with hydrogen as electron donor. The genome of DvH revealed the presence of cydAB and cox genes, encoding a quinol oxidase bd and a cytochrome c oxidase, respectively. In the membranes of DvH, we detected both quinol oxygen reductase [inhibited by heptyl-hydroxyquinoline-N-oxide (HQNO)] and cytochrome c oxidase activities. Spectral and HPLC data for the membrane fraction revealed the presence of o-, b- and d-type haems, in addition to a majority of c-type haems, but no a-type haem, in agreement with carbon monoxide-binding analysis. The cytochrome c oxidase is thus of the cc(o/b)o3 type, a type not previously described. The monohaem cytochrome c553 is an electron donor to the cytochrome c oxidase; its encoding gene is located upstream of the cox operon and is 50-fold more transcribed than coxI encoding the cytochrome c oxidase subunit I. Even when DvH is grown under anaerobic conditions in lactate/sulfate medium, the two terminal oxidase-encoding genes are expressed. Furthermore, the quinol oxidase bd-encoding genes are more highly expressed than the cox genes. The cox operon exhibits an atypical genomic organization, with the gene coxII located downstream of coxIV. The occurrence of these membrane-bound oxygen reductases in other strictly anaerobic Deltaproteobacteria is discussed.


Subject(s)
Desulfovibrio vulgaris/enzymology , Membrane Proteins/metabolism , Oxidoreductases/metabolism , Oxygen/metabolism , Cell Membrane/metabolism , Desulfovibrio vulgaris/genetics , Electrons , Enzyme Activation , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Gene Order , Operon , Oxidation-Reduction , Phylogeny
4.
Appl Microbiol Biotechnol ; 60(3): 352-60, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12436319

ABSTRACT

Developing new bioremediation processes for soils and effluents polluted by Cr(VI) requires the selection of the most efficient and the most heavy-metal-resistant bacteria. The effects of Cr(VI) on bioenergetic metabolism in two sulfate-reducing bacteria (SRB), Desulfovibrio vulgaris Hildenborough and Desulfomicrobium norvegicum, were monitored using isothermal microcalorimetry. The complete reduction of Cr(VI) to Cr(III) was studied by spectrophotometry and by speciation using a combination of high-performance liquid chromatography and inductively coupled plasma-mass spectrometry. Results revealed that Cr(VI) induces an inhibition of growth with concomitant production of energy, which can be compared to the reaction of the bacteria to a stress such as oxidative stress. Moreover, the sensitivity of bacteria towards this metal is as a characteristic of the strain, which leads to differences in the kinetics of Cr(VI) reduction. The study by microcalorimetry of heavy metal effects on SRB bioenergetic metabolism thus appears an appropriate tool to identify better strains to be used for industrial bioremediation process development.


Subject(s)
Chromates/metabolism , Sulfur-Reducing Bacteria/metabolism , Thermodynamics , Biodegradation, Environmental , Chromates/pharmacology , Oxidation-Reduction , Sulfur-Reducing Bacteria/drug effects
5.
Arch Microbiol ; 174(3): 143-51, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11041344

ABSTRACT

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a transmembrane redox protein complex (the Hmc complex) that has been proposed to catalyze electron transport linking periplasmic hydrogen oxidation to cytoplasmic sulfate reduction. We have replaced a 5-kb DNA fragment containing most of the hmc operon by the cat gene. The resulting chloramphenicol-resistant mutant D. vulgaris H801 grows normally when lactate or pyruvate serve as electron donors for sulfate reduction. Growth with hydrogen as electron donor for sulfate reduction (acetate and CO2 as the carbon source) is impaired. These results confirm the importance of the Hmc complex in electron transport from hydrogen to sulfate. Mutant H801 is also deficient in low-redox-potential niche establishment. On plates, colony development takes 14 days longer than colony development of the wild-type strain, when the cells use hydrogen as the electron donor. This result suggests that, in addition to transmembrane electron transport from hydrogen to sulfate, the redox reactions catalyzed by the Hmc complex are crucial in establishment of the required low-redox-potential niche that allows single cells to grow into colonies.


Subject(s)
Bacterial Proteins/genetics , Desulfovibrio vulgaris/metabolism , Gene Deletion , Hydrogen/metabolism , Operon , Bacterial Proteins/metabolism , Chloramphenicol Resistance/genetics , Culture Media , Desulfovibrio vulgaris/genetics , Desulfovibrio vulgaris/growth & development , Electron Transport/genetics , Electrophoresis, Polyacrylamide Gel/methods , Genes, Bacterial , Hydrogenase/metabolism , Immunoblotting , Oxidation-Reduction , Phenotype , Sulfates/metabolism
6.
Biochim Biophys Acta ; 1481(1): 18-24, 2000 Aug 31.
Article in English | MEDLINE | ID: mdl-11004576

ABSTRACT

The production of Desulfovibrio vulgaris Hildenborough cytochrome c(3) (M(r) 13000), which is a tetraheme cytochrome, in Escherichia coli was examined. This cytochrome was successfully produced in an E. coli strain co-expressing the ccmABCDEFGH genes involved in the cytochrome c maturation process. The apocytochrome c(3) was matured in either anaerobic or aerobic conditions, but aerobic growth in the presence of delta-aminolevulinic acid was found to be best for cytochrome c(3) production. Site-directed mutagenesis was performed to investigate the effect of the presence of four amino acids in between the two cysteines of the heme binding sites 2 and 4 on the maturation of holocytochrome c(3) in E. coli.


Subject(s)
Cytochrome c Group/genetics , Escherichia coli/genetics , Aminolevulinic Acid/pharmacology , Binding Sites , Cytochrome c Group/biosynthesis , Desulfovibrio vulgaris/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Heme/chemistry , Mutagenesis, Site-Directed , Mutation , Periplasm/enzymology , Plasmids
7.
Biochemistry ; 39(10): 2530-7, 2000 Mar 14.
Article in English | MEDLINE | ID: mdl-10704202

ABSTRACT

The combination of docking algorithms with NMR data has been developed extensively for the studies of protein-ligand interactions. However, to extend this development for the studies of protein-protein interactions, the intermolecular NOE constraints, which are needed, are more difficult to access. In the present work, we describe a new approach that combines an ab initio docking calculation and the mapping of an interaction site using chemical shift variation analysis. The cytochrome c553-ferredoxin complex is used as a model of numerous electron-transfer complexes. The 15N-labeling of both molecules has been obtained, and the mapping of the interacting site on each partner, respectively, has been done using HSQC experiments. 1H and 15N chemical shift analysis defines the area of both molecules involved in the recognition interface. Models of the complex were generated by an ab initio docking software, the BiGGER program (bimolecular complex generation with global evaluation and ranking). This program generates a population of protein-protein docked geometries ranked by a scoring function, combining relevant stabilization parameters such as geometric complementarity surfaces, electrostatic interactions, desolvation energy, and pairwise affinities of amino acid side chains. We have implemented a new module that includes experimental input (here, NMR mapping of the interacting site) as a filter to select the accurate models. Final structures were energy minimized using the X-PLOR software and then analyzed. The best solution has an interface area (1037.4 A2) falling close to the range of generally observed recognition interfaces, with a distance of 10.0 A between the redox centers.


Subject(s)
Cytochrome c Group/chemistry , Ferredoxins/chemistry , Models, Molecular , Amino Acid Sequence , Base Sequence , Computer Simulation , Cytochrome c Group/genetics , Cytochrome c Group/metabolism , Desulfovibrio vulgaris/enzymology , Electron Transport , Ferredoxins/genetics , Ferredoxins/metabolism , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular/methods , Peptide Mapping , Sulfur-Reducing Bacteria/chemistry
8.
Appl Environ Microbiol ; 66(2): 671-7, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10653734

ABSTRACT

To explore the physiological role of tetraheme cytochrome c(3) in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion. The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful. The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities. Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant. Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium. These data support a role for tetraheme cytochrome c(3) in the electron transport pathway from pyruvate to sulfate or sulfite in D. desulfuricans G20.


Subject(s)
Cytochrome c Group/genetics , Cytochrome c Group/metabolism , Desulfovibrio/genetics , Desulfovibrio/metabolism , Mutation , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Conjugation, Genetic , Desulfovibrio/growth & development , Molecular Sequence Data , Periplasm/metabolism , Plasmids/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Transcription, Genetic
9.
Biochim Biophys Acta ; 1476(1): 85-92, 2000 Jan 03.
Article in English | MEDLINE | ID: mdl-10606770

ABSTRACT

A central step in the energy metabolism of sulfate-reducing bacteria is the oxidation of molecular hydrogen, catalyzed by a periplasmic hydrogenase. The resulting electrons are then transferred to various electron transport chains and used for cytoplasmic sulfate reduction. The complex formation between [NiFeSe] hydrogenase and the soluble periplasmic polyheme cytochromes from Desulfomicrobium norvegicum was characterized by cross-linking experiments, BIAcore and kinetics analysis. Analysis of electron transfer between [NiFeSe] hydrogenase and octaheme cytochrome c(3) (M(r) 26¿ omitted¿000) pointed out that this cytochrome is reduced faster in the presence of catalytic amounts of tetraheme cytochrome c(3) (M(r) 13¿ omitted¿000) isolated from the same organism. The activation of the hydrogenase-dependent reduction of polyheme cytochromes by cytochrome c(3) (M(r) 13¿ omitted¿000), which is now described in both Desulfovibrio and Desulfomicrobium, is proposed as a general mechanism. During this process, cytochrome c(3) (M(r) 13¿ omitted¿000) would act as an electron shuttle in between hydrogenase and the polyheme cytochromes and its conductivity appears to be an important factor.


Subject(s)
Cytochromes/chemistry , Hydrogenase/chemistry , Sulfur-Reducing Bacteria/enzymology , Cytochrome c Group/chemistry , Electron Transport , Electrophoresis, Polyacrylamide Gel , Energy Metabolism , Enzyme Activation , Kinetics , Oxidation-Reduction
10.
Eur J Biochem ; 261(2): 398-404, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10215849

ABSTRACT

When using heteronuclear NMR, 15N-labelling is necessary for structural analysis, dynamic studies and determination of complex formation. The problems that arise with isotopic labelling of metalloproteins are due to their complex maturation process, which involves a large number of factors. Cytochromes c are poorly expressed in Escherichia coli and the overexpression that is necessary for 15N-labelling, requires an investigation of the expression host and special attention to growth conditions. We have succeeded in the heterologous expression and the complete and uniform isotopic 15N-labelling of the cytochrome c553 from Desulfovibrio vulgaris Hildenborough, in a sulphate-reducing bacterium, D. desulfuricans G200, by using a growth medium combining 15N-ammonium chloride and 15N-Celtone. These conditions allowed us to obtain approximately 0.8 mg x L-1 of pure labelled cytochrome c553. 1H and 15N-assignments for both the oxidized and the reduced states of cytochrome c553 were obtained from two-dimensional heteronuclear experiments. Pseudocontact effects due to the haem Fe3+ have been analysed for the first time through 15N and 1H chemical shifts in a c-type cytochrome.


Subject(s)
Cytochrome c Group/chemistry , Desulfovibrio vulgaris/chemistry , Cytochrome c Group/biosynthesis , Cytochrome c Group/isolation & purification , Heme/chemistry , Isotope Labeling , Magnetic Resonance Spectroscopy , Nitrogen Isotopes , Oxidation-Reduction , Protein Structure, Secondary
11.
Biochemistry ; 38(1): 33-41, 1999 Jan 05.
Article in English | MEDLINE | ID: mdl-9890880

ABSTRACT

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.


Subject(s)
Cytochrome c Group/chemistry , Phenylalanine/chemistry , Phenylalanine/physiology , Amino Acid Substitution , Calorimetry, Differential Scanning , Circular Dichroism , Computer Simulation , Cytochrome c Group/isolation & purification , Cytochrome c Group/metabolism , Desulfovibrio vulgaris/enzymology , Electron Transport , Hydrogenase/metabolism , Models, Molecular , Phenylalanine/metabolism , Structure-Activity Relationship
12.
Eur J Biochem ; 253(3): 645-52, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9654061

ABSTRACT

The electron transfer between formate dehydrogenase and cytochrome c553 from the anaerobic bacteria Desulfovibrio vulgaris Hildenborough has been investigated. Parameters of the electron transfer kinetics are reported. The ionic strength dependence of the complex formation has been evidenced. Two mutants of cytochrome c553 have been obtained using site-directed mutagenesis with the substitutions K62E and K62E,K63E. According to one-dimensional and two-dimensional NMR analysis, the two variants were found to have the same folding pattern as that of the wild-type cytochrome. The replacements of the lysine residues by acidic groups have important effects on the affinity between the two oxidoreduction partners. K62 and K63 are essential for recognition between the formate dehydrogenase and the cytochrome c553. Previous structural studies of cytochrome c553 have demonstrated the involvement of the polypeptide chain in the modulation of the particular low oxidoreduction potential of this cytochrome. The present study provides evidence that, during the evolution of cytochromes from the anaerobic metabolism to aerobic respiration and photosynthesis, the electrostatic distribution at the recognised encounter surface around the heme is highly conserved in all cytochromes.


Subject(s)
Cytochrome c Group/chemistry , Cytochrome c Group/metabolism , Desulfovibrio vulgaris/metabolism , Formate Dehydrogenases/chemistry , Formate Dehydrogenases/metabolism , Protein Conformation , Amino Acid Sequence , Amino Acid Substitution , Animals , Cytochrome c Group/isolation & purification , Electron Transport , Formate Dehydrogenases/isolation & purification , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Tuna
13.
Biochemistry ; 37(23): 8331-40, 1998 Jun 09.
Article in English | MEDLINE | ID: mdl-9622485

ABSTRACT

Replacement of tyrosine 64 by alanine in cytochrome c553 from Desulfovibrio vulgarisHildenborough prevents electron transfer with the formate dehydrogenase. Biophysical and biochemical studies show that the protein is correctly folded and that the oxidoreduction potential is not modified. The solution structure of the mutant cytochrome determined by two-dimensional (2D) NMR clearly establishes that the overall fold of the molecule is nearly identical to that of the wild-type cytochrome. The electrostatic surface charge distributions for the wild-type and mutant cytochrome are similar, suggesting that the interaction site of the physiological partners is not modified by the mutation. The lack of the aromatic ring induces slight destabilization of the hydrophobic core of the molecule and modifications of the hydrogen bond at position 64, as well as conformational disorder of the side chain of K63. The loss of the hydrogen bond from tyrosine 64 and the increase of the solvent exposure of the heme are probably responsible of the loss of electron transfer between formate dehydrogenase and cytochrome c553.


Subject(s)
Cytochrome c Group/metabolism , Desulfovibrio vulgaris/enzymology , Formate Dehydrogenases/metabolism , Tyrosine/metabolism , Amino Acid Substitution/genetics , Arginine/genetics , Cytochrome c Group/chemistry , Cytochrome c Group/genetics , Electron Transport/genetics , Formate Dehydrogenases/chemistry , Heme/chemistry , Hydrogen Bonding , Kinetics , Magnetic Resonance Spectroscopy , Models, Molecular , Mutagenesis, Site-Directed , Oxidation-Reduction , Protein Conformation , Static Electricity , Tyrosine/chemistry , Tyrosine/genetics
14.
Appl Environ Microbiol ; 64(4): 1308-12, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9546165

ABSTRACT

Multiheme cytochrome c proteins that belong to class III have been recently shown to exhibit a metal reductase activity, which could be of great environmental interest, especially in metal bioremediation. To get a better understanding of these activities, the gene encoding cytochrome c7 from the sulfur-reducing bacterium Desulfuromonas acetoxidans was cloned from genomic DNA by PCR and expressed in Desulfovibrio desulfuricans G201. The expression system was based on the cyc transcription unit from Desulfovibrio vulgaris Hildenborough and led to the synthesis of holocytochrome c7 when transferred by electrotransformation into the sulfate reducer Desulfovibrio desulfuricans G201. The produced cytochrome was indistinguishable from the protein purified from Desulfuromonas acetoxidans cells with respect to several biochemical and biophysical criteria and exhibited the same metal reductase activities as determined from electrochemical experiments. This suggests that the molecule was correctly folded in the host organism. Desulfovibrio desulfuricans produces functional multiheme c-type cytochromes from bacteria belonging to a different genus and may be considered a suitable host for the heterologous biogenesis of multiheme c-type cytochromes for either structural or engineering studies. This report, which presents the first example of the transformation of a Desulfovibrio desulfuricans strain by electrotransformation, describes work that is the first necessary step of a protein engineering program that aims to specify the structural features that are responsible for the metal reductase activities of multiheme cytochrome c7.


Subject(s)
Cytochrome c Group/genetics , Cytochrome c Group/metabolism , Desulfovibrio/genetics , Desulfovibrio/metabolism , Metals/metabolism , Sulfur-Reducing Bacteria/genetics , Sulfur-Reducing Bacteria/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cytochrome c Group/chemistry , DNA, Bacterial/genetics , DNA, Recombinant/genetics , Genes, Bacterial , Molecular Sequence Data , Oligodeoxyribonucleotides/genetics , Oxidoreductases/chemistry , Oxidoreductases/genetics , Oxidoreductases/metabolism , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
15.
Biochemistry ; 37(8): 2120-30, 1998 Feb 24.
Article in English | MEDLINE | ID: mdl-9485359

ABSTRACT

A combination of structural, kinetic, and interaction experiments has been used to study the role of a highly conserved aromatic residue, Tyr73, parallel to the sixth heme axial ligand of heme 4 in multiheme cytochrome c3 (Mr = 26 000), also called cytochrome cc3 or octaheme cytochrome, from Desulfovibrio desulfuricans Norway. This residue is expected to be involved in intermolecular electron transfer and protein-protein interaction, since heme 4 is described to be the interaction site between physiological partners. The kinetic experiments show that the Y73E replacement provokes no significant change in the electron-transfer reaction with the physiological partner, the [NiFeSe] hydrogenase, but that the protein-protein interaction between cytochrome c3 (Mr = 26 000) and hydrogenase is strongly affected by the mutation. The aromatic residue does not play a role in maintaining the axial heme ligand in a particular orientation, since the mutation did not affect the orientation of histidine 77, the sixth axial ligand of heme 4. The structural analysis by X-ray crystallography clearly shows that a rearrangement of the charged residues in the vicinity of the mutation site is responsible for the change in protein-protein interaction, which is of an electrostatic nature. Lys22 and Arg66, residues which are located at the interacting surface, are twisted toward the mutated position Glu73 in order to compensate for the negative charge and therefore are no longer accessible for the docking with a physiological partner. Tyr73 has instead a structural function and probably a role in maintaining the hydrophobic environment of the heme 4 cavity rather than a function in the intermolecular electron transfer with the physiological partners.


Subject(s)
Cytochrome c Group/chemistry , Cytochrome c Group/genetics , Desulfovibrio/enzymology , Binding Sites/genetics , Cytochrome c Group/metabolism , Desulfovibrio/genetics , Electrochemistry , Electron Transport , Heme/chemistry , Kinetics , Models, Molecular , Molecular Sequence Data , Molecular Weight , Oxidation-Reduction , Point Mutation , Protein Conformation
16.
Eur J Biochem ; 251(3): 787-94, 1998 Feb 01.
Article in English | MEDLINE | ID: mdl-9490053

ABSTRACT

Y64 has been replaced in cytochrome c553 from Desulfovibrio vulgaris Hildenborough by phenylalanine, leucine, valine, serine and alanine residues. An NMR study of structural variation induced in both oxidoreduction states of the molecule has been carried out by analysing observed chemical-shift variations. Dynamic changes were evidenced using NH exchange. We have observed that the substitution has a drastic effect on the stability of the molecule in the reduced state, although there is no effect on the reduction potential of the cytochrome. Y64-->F substitution induces particular effects on the NH exchange at the N-terminal, C-terminal and central alpha-helices and increases the stability of the oxidized molecule.


Subject(s)
Cytochrome c Group/chemistry , Desulfovibrio vulgaris/metabolism , Protein Conformation , Amino Acid Sequence , Amino Acid Substitution , Conserved Sequence , Hydrogen , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular/methods , Oxidation-Reduction , Protein Folding , Recombinant Proteins/chemistry
17.
Biochem Biophys Res Commun ; 242(1): 213-8, 1998 Jan 06.
Article in English | MEDLINE | ID: mdl-9439638

ABSTRACT

A monoheme cytochrome c553 and a hexadecaheme high molecular weight cytochrome (Hmc) have been isolated and characterized from the sulfate-reducing bacteria Desulfovibrio desulfuricans G201, in addition to the tetraheme cytochrome c3 (Mr 13000) that has been previously described. Both cytochromes are homologous with respect to several biochemical properties to the corresponding cytochromes found in other Desulfovibrio species. However, they are acidic proteins while the corresponding molecules, isolated from other Desulfovibrio species, are relatively more basic. The D. desulfuricans cytochrome content appears identical to that of D. vulgaris Hildenborough. Isolation of these cytochromes from a Desulfovibrio desulfuricans strain is of great interest in order to get more insight on the physiological function of these molecules.


Subject(s)
Cytochrome c Group/analysis , Desulfovibrio/chemistry , Amino Acid Sequence , Bacterial Proteins/analysis , Molecular Sequence Data , Molecular Weight , Sequence Homology, Amino Acid
18.
J Biol Chem ; 272(24): 15128-34, 1997 Jun 13.
Article in English | MEDLINE | ID: mdl-9182533

ABSTRACT

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.


Subject(s)
Cytochrome c Group/metabolism , Desulfovibrio/genetics , Heme/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cytochrome c Group/genetics , DNA, Bacterial , Desulfovibrio/enzymology , Molecular Sequence Data , Molecular Weight , Mutagenesis, Site-Directed
19.
Biochem J ; 320 ( Pt 3): 933-8, 1996 Dec 15.
Article in English | MEDLINE | ID: mdl-9003383

ABSTRACT

Cytochrome c3 (M(r) 26000) isolated from Desulfovibrio gigas is a dimeric cytochrome consisting of two identical subunits of 109 amino acids, each of which contains four haem groups. On the basis of its amino acid sequence, this cytochrome clearly belongs to the cytochrome c3 superfamily, and will be classified in class III of the c-type cytochromes as defined by Ambler [(1980) in From Cyclotrons to Cytochromes (Robinson, A. B. and Kaplan, N. O., eds.), pp. 263-279, Academic Press, London]. It contains ten cysteine and nine histidine residues in each subunit, and eight cysteines and eight histidines linked to the four haem groups were found to be invariant on alignment of all known cytochrome c3 sequences. Two intermolecular disulphide bridges have been determined between cysteine residues 5 and 46 of the two monomers. Cytochrome c3 (M(r) 26,000) from D gigas is clearly different from cytochrome c3 (M(r) 13,000) from the same strain, with which it shows only 27% sequence identity. Compared with cytochrome c3 (M(r) 26,000) from D. desulfuricans Norway, the three-dimensional structure of which has been determined, 26.95% of the residues have been conserved. In the enzyme from D. desulfuricans Norway, hydrophobic interactions have been described across the dimer interface. Residues involved in similar interactions seem to be well conserved in the equivalent D. gigas cytochrome. This sequence provides structural data to allow specification of this new subclass of polyhaem cytochromes. Furthermore, D. gigas cytochrome c3 (M(r) 26,000) is the first polyhaem cytochrome shown to contain two disulphide bridges linking two identical subunits, which could induce more rigid folding. The folding and the evolution of this family of polyhaem cytochromes are discussed.


Subject(s)
Cytochrome c Group/chemistry , Desulfovibrio/chemistry , Amino Acid Sequence , Amino Acids/analysis , Dimerization , Disulfides/chemistry , Electron Transport , Heme/analysis , Iron/analysis , Kinetics , Molecular Sequence Data , Molecular Weight , Peptide Fragments/analysis , Peptide Fragments/chemistry , Protein Conformation , Protein Denaturation , Protein Folding , Sequence Alignment , Sequence Analysis
20.
FEBS Lett ; 395(1): 53-7, 1996 Oct 14.
Article in English | MEDLINE | ID: mdl-8849688

ABSTRACT

In order to study the conformational stability induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough (DvH) cytochrome c553, fast peptic digestion of deuterated protein followed by separation and measurement of related peptides using liquid chromatography coupled to electrospray ionization mass spectrometry was performed. We show that the H-bonding and/or solvent accessibility properties were modified by the single-site mutation. The mutant proteins can be classified into two groups: the Y64F and Y64L mutants with nearly unchanged deuterium incorporation compared to the wild-type protein and the Y64S, Y64V and Y64A mutants with increased deuterium incorporation. The 70-74 peptide was the most affected by mutation of Tyr-64, the phenylalanine mutant inducing slight stabilization whereas the serine mutant was significantly destabilized. In addition, from the analysis of the overlapping 37-57 and 38-57 peptides we can conclude that the amide proton of Tyr-38 has been replaced by deuterium in all proteins.


Subject(s)
Cytochrome c Group/chemistry , Desulfovibrio vulgaris/chemistry , Deuterium , Protein Conformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Tyrosine/physiology , Electrons , Hydrogen Bonding , Molecular Weight , Mutation , Oxidation-Reduction , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Solvents
SELECTION OF CITATIONS
SEARCH DETAIL
...