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Biomedical subjects

J Gaillard

Publications and source records attributed to J Gaillard.

At least 37 records · Page 2Linked to original sources
PubMed1998

The effects of ultraviolet radiation on antibiotic-resistant bacteria in vitro.

Wound infections produced by antibiotic-resistant bacterial strains are particularly difficult to manage. This study examined the effectiveness of ultraviolet (UV) light treatment in killing antibiotic-resistant strains of Staphylococcus aureus and Enterococcus faecalis in vitro. Between 2 and 5 replications of each organism at 10(8) organisms/ml were prepared and plated on sheep blood agar medium and treated with UV light (254 nm, 15.54 mW/cm2 output). Irradiation times were 0, 2, 5, 8, 15, 30, 45, 60, 90 or 120 seconds. Bacterial cultures were then incubated at 35 degrees C for 24 hours. Kill rates were 99.9 percent for the methicillin-resistant strain of S. aureus (MRSA) at 5, 8, 15, 30, 45, 60 seconds and 100 percent at 90 and 120 seconds. Kill rates were 99.9 percent at 5, 8, 15, 30 seconds for vancomycin-resistant E. faecalis (VRE) and 100 percent at 45, 60, 90, 120 seconds. Similar results were found with UV light treatment of the antibiotic-susceptible strains of S. aureus and E. faecalis. A significant difference in kill rates at 30 seconds of UV exposure was detected between the antibiotic-resistant strain of S. aureus and the antibiotic-resistant strain of E. faecalis (Student's t test, p < 0.01). Significant differences were also detected in the kill rates at 30 second exposure times for the antibiotic-susceptible strains of S. aureus and E. faecalis. These findings suggest that the Enterococcal bacteria is more susceptible to the killing effects of UV. This data also suggests that UV light at 254 nm is bactericidal for antibiotic-resistant strains of S. aureus and E. faecalis at times as short as 5 seconds and that the enterococcal bacteria is more susceptible to the killing effects of UV. With recommended patient treatment times for infected wounds being significantly longer than 5 seconds, this data indicates that patient treatment times need to be re-examined.

Cross Infection↗
PubMed1997

Site-directed mutagenesis of rubredoxin reveals the molecular basis of its electron transfer properties.

Rubredoxins contain a single non-heme iron atom coordinated by four cysteines. This iron is redox active and confers a role to these proteins in electron transfer chains. The structural features responsible for setting the values of the reduction potential and of the electron self-exchange rate constant have been probed by site-directed mutagenesis. Replacements of the highly conserved residues in positions 8, 10, and 11 (valine, glycine, and tyrosine, respectively) all lead to shifts of the reduction potential, up to 75 mV. These cannot be explained by simple considerations about the physicochemical properties of the substituting side chains but rather indicate that the value of the reduction potential is finely tuned by a variety of interactions. In contrast, the electron self exchange rate constant measured by nuclear magnetic resonance does not vary much, except when a charged residue is included in position 8 or 10, at the surface of the protein closest to the iron atom. Analysis of the data with a model for electrostatic interactions, including both monopolar and dipolar terms, indicates that the presence of a charge in this region not only increases the repulsion between molecules but also affects the electron transfer efficiency of the bimolecular complexes formed. The studies presented constitute a first step toward probing the structural elements modulating the reactivity of the FeS4 unit in a protein and defining the electron transfer active site(s) of rubredoxin.

Binding Sites↗
PubMed1997

Assembly of a [2Fe-2S]2+ cluster in a molecular variant of Clostridium pasteurianum rubredoxin.

The rubredoxin from Clostridium pasteurianum contains a single iron atom bound to the polypeptide chain by cysteines 6, 9, 39, and 42. The C42A variant of this protein has been prepared by site-directed mutagenesis and heterologous expression of the gene in Escherichia coli. The mutated protein was found to contain an unexpected chromophore that has been characterized by a variety of techniques. UV-visible absorption and resonance Raman spectra were strongly reminiscent of those of [2Fe-2S] proteins. Mössbauer spectra of the oxidized chromophore isolated in oxygen-free conditions indicated low-temperature diamagnetism resulting from antiferromagnetically coupled high-spin ferric ions. Analysis of X-ray absorption fine structure spectra yielded an Fe-Fe distance of 2.68 A. Colorimetric assays of iron and inorganic sulfide showed that the two elements are present in a 1:1 ratio. Electrospray-ionization mass spectra displayed a major component at M = 6190 Da, i.e. the molecular mass of the C42A apoprotein plus two atomic masses of iron and two atomic masses of sulfur. Taken together, these data show that a mere point mutation allows the stabilization of a binuclear [2Fe-2S] cluster in a protein that normally accommodates a mononuclear Fe(Scys)4 site. Assembly of a [2Fe-2S] cluster may occur because rubredoxin assumes a similar fold around its metal center as the [2Fe-2S] Rieske protein. Alternatively, a more extensive structural rearrangement of the polypeptide chain of the C42A rubredoxin variant may be considered as well.

Binding Sites↗
PubMed1997

Activation of the anaerobic ribonucleotide reductase from Escherichia coli. The essential role of the iron-sulfur center for S-adenosylmethionine reduction.

The anaerobic ribonucleotide reductase of Escherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an alpha2beta2 heterotetramer. In its active form, the large alpha2 subunit contains an oxygen-sensitive glycyl radical, whereas the beta2 small protein harbors a [4Fe-4S] cluster that joins its two polypeptide chains. Formation of the glycyl radical in the inactive enzyme requires S-adenosylmethionine (AdoMet), dithiothreitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (dithionite or 5-deazaflavin plus light) reducing system. Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of beta2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. Direct binding experiments showed that AdoMet binds to beta2 with a Kd of 10 microM and a 1:1 stoichiometry. Binding was confirmed by EPR spectroscopy that demonstrated the formation of a complex between AdoMet and the [4Fe-4S] center of beta2. Dithiothreitol triggered the cleavage of AdoMet, leading to an EPR-silent form of beta2 and, in the case of alpha2beta2, to glycyl radical formation. In both instances, 3 methionines were formed per mol of protein. Our results indicate that the Fe-S center of beta2 is directly involved in the reductive cleavage of AdoMet and suggest a new biological function for an iron-sulfur center, i.e redox catalysis, as recently proposed by others (Staples, R. C., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A. L., Schürmann, P., Knaff, D. B., and Johnson, M. K. (1996) Biochemistry 35, 11425-11434).

Catalysis↗
PubMed1997

EPR investigation of compound I in Proteus mirabilis and bovine liver catalases: formation of porphyrin and tyrosyl radical intermediates.

Compound I of Proteus mirabilis and bovine liver catalases (PMC and BLC, respectively) were studied combining EPR spectroscopy and the rapid-mix freeze-quench techniques. Both enzymes, when treated with peroxyacetic acid, form a catalytic intermediate which consists of an oxoferryl porphyrin pi-cation radical. In PMC this intermediate is semistable, and an unexpected reversible equilibrium under pH influence takes place between two forms of compound I with different coupling between the oxoferryl and the porphyrin pi-cation radical. At acid pH, one form has a ferromagnetic character as in Micrococcus luteus compound I. At neutral pH, another form with a much smaller coupling, reminiscent of the horse radish peroxidase compound I, is detected. The approximate midpoint, estimated for these changes in the range 5.3 < pH < 6.0, approaches the pKa value of an histidyl residue. The residues possibly involved in the transformation are discussed in terms of the known structure of PMC compound I. The EPR spectrum of BLC compound I (pH 5.6), obtained in the millisecond time scale (40 ms), also showed a mixture of two forms which, most probably, correspond to two different magnetic exchange interactions, as in the case of PMC. Taken together, the low-temperature electronic absorption and the EPR spectra of BLC compound I formed in the 0.04-15 s range show that the porphyrin pi-cation radical disappears and, instead, a tyrosyl radical is formed. ENDOR experiments confirm our previously estimated hyperfine couplings to the C2,6 and C3,5 ring protons and the beta-methylene protons of the purported tyrosyl radical. Candidates for such a tyrosyl radical are discussed in connection with the possible electron transfer pathways between the heme active site and the NADPH cofactor.

Animals↗
PubMed1997

Intramolecular electron transfer between [4Fe-4S] clusters studied by proton magnetic resonance spectroscopy.

The rate constants for the intramolecular electron transfer between the two [4Fe-4S] clusters of a series of native and genetically engineered ferredoxins have been determined by proton magnetic resonance (1H NMR) spectroscopy. The measurement relies on the properties of the signals assigned to beta-protons of the coordinating cysteines when the protein is substoichiometrically reduced: these signals include coalesced peaks arising from the fast hopping of an extra electron between the two oxidized clusters of the protein. An upper limit of significantly less than 10(5) M(-1) s(-1) for the intermolecular and an average of the order of 5 x 10(6) s(-1) for the intramolecular electron transfer rate constants of several ferredoxins have been obtained. Owing to the edge-to-edge intercluster distance of approximately 10 A derived from the crystallographic structure of Clostridium acidurici ferredoxin, the rate constant associated with the intramolecular process is as expected for a nonadiabatic redox process, assuming a reasonable value of less than 1 eV for the reorganization energy. The latter could not be determined from the temperature dependence of the rate constant since no variation was observed over the temperature range accessible in these experiments. Structural changes introduced around and between the two [4Fe-4S] clusters in Clostridium pasteurianum ferredoxin by site-directed mutagenesis have been used to probe the potential involvement of dominant electron transfer pathways between the clusters. These changes have no major effect on the value of the intramolecular electron transfer rate constant. From this analysis, no specific amino acid side chain seems to play a central role in the process. The rate constants derived in the present work may serve as a basis for the study of enzymes containing two closely spaced [4Fe-4S] clusters such as found in these ferredoxins.

Electrons↗
PubMed1996

Electron transfer across the O2- generating flavocytochrome b of neutrophils. Evidence for a transition from a low-spin state to a high-spin state of the heme iron component.

The NADPH oxidase complex of activated neutrophils consists of a membrane-bound flavocytochrome b and cytosolic activation factors. Despite its ability to react with O2, the heme b component of the flavocytochrome is insensitive to cyanide and CO2, and slowly reactive to butyl isocyanide. We report here that arachidonic acid, an anionic amphophil which elicits oxidase activation in a cell-free system induces a transition of the heme iron of the neutrophil flavocytochrome b from a low-spin hexacoordinated state to a high-spin pentacoordinated state and promotes the binding of butyl isocyanide to the heme b. Low-temperature EPR spectra of air-oxidized flavocytochrome b either purified or in its membrane-bound form showed a low-spin signal at g = 3.26 and a high-spin signal at g = 6.0. Upon addition of arachidonic acid, the g = 3.26 signal vanished; a low-spin signal at g = 2.23 appeared, and the signal at g = 6.0 progressively increased. The subsequent addition of butyl isocyanide resulted in the decrease of the g = 6.0 and g = 2.23 signals and in the appearance of a new low-spin signal at g = 2.33. Consistent with the EPR results, upon addition of arachidonic acid to oxidized flavocytochrome b, a 2.5 nm blue shift of the Soret peak was detected in low-temperature optical spectra. The subsequent addition of butyl isocyanide resulted in the emergence of a peak at 432 nm reflecting the formation of a butyl isocyanide-oxidized heme b complex. In the case of sodium dithionite-reduced flavocytochrome b, arachidonic acid promoted the binding of butyl isocyanide to the reduced heme b, as shown by the emergence of a peak at 434 nm and the decrease of the alpha band at 558 nm. The same promoting effect was encountered with sodium dodecyl sulfate, an anionic amphophil capable of eliciting oxidase activation like arachidonic acid. In contrast to arachidonic acid, arachidonic acid methyl ester was ineffective and counteracted the effect of arachidonic acid. Butyl isocyanide added to intact neutrophils was found to bind to heme b, only after the cells have been activated. These data demonstrate the transient accumulation of a pentacoordinated form of the heme iron of flavocytochrome b under in vitro and in vivo conditions; the pentacoordinated form of the reduced heme b is postulated to react with O2 to generate the superoxide anion.

Animals↗
PubMed1996

Inhibition by iodoacetamide and acetylene of the H-D-exchange reaction catalyzed by Thiocapsa roseopersicina hydrogenase.

The kinetics of H-D isotope exchange catalyzed by the thermostable hydrogenase from Thiocapsa roseopersicina have been studied by analysis of the exchange between D2 and H2O. The pH dependence of the exchange reaction was examined between pH 2.5 and pH 11. Over the whole pH range, HD was produced at a higher initial velocity than H2, with a marked optimum at pH 5.5; a second peak in the pH profile was observed at around pH 8.5. The rapid formation of H2 with respect to HD in the D2/H2O system is consistent with a heterolytic cleavage of D2 into D+ and an enzyme hydride that can both exchange with the solvent. The H-D-exchange activity was lower in the H2/D2O system than in the D2/H2O system. The other reactions catalyzed by the hydrogenase, H2 oxidation and H2 evolution, are pH dependent; the optimal pH were 9.5 for H2 uptake and 4.0 for H2 production. Treatment of the active form of hydrogenase by iodoacetamide led to a slow and irreversible inhibition of the H-D exchange. When iodo[1-14C]acetamide was incubated with hydrogenase, the radioactive labeling of the large subunit was higher for the enzyme activated under H2 than for the inactive oxidized form. Cysteine residues were identified as the alkylated derivative by amino acid analysis. Acetylene, which inhibits H-D exchange and abolishes the Ni-C EPR signal, protected the enzyme from irreversible inhibition by iodoacetamide. These data indicate that iodoacetamide can reach the active site of the H2-activated hydrogenase from T. roseopersicina. This was not found to be the case with the seleno hydrogenase from Desulfovibrio baculatus (now Desulfomicrobium baculatus). Cysteine modification by iodoacetamide upon activation of the enzyme concomitant with loss of H-D exchange indicates that reductive activation makes at least one Cys residue of the active site available for alkylation.

Acetylene↗
PubMed1996

Use of 1H longitudinal relaxation times in the solution structure of paramagnetic proteins. Application to [4Fe-4S] proteins.

The accuracy of the solution structures determined by NMR is often poor around paramagnetic centers because the properties of the near protons are strongly perturbed by the electronic spin. The structural information contained in the relaxation rates of these protons has been extracted here by measuring the longitudinal relaxation times with the inversion-recovery total correlation spectroscopy (IR-TOCSY) sequence based on the recovery of cross peaks. In addition to measurements with nonselective inversion-recovery for nonoverlapping signals, reliable data have been obtained for a majority of main-chain protons from Chromatium vinosum high-potential ferredoxin. When a small and constant contribution from diamagnetism as well as the electronic spin distribution over the [4Fe-4S] cluster are taken into account, the shortest longitudinal relaxation times depend directly on the distance separating the protons from the paramagnetic center. This indicates that electron-nuclei dipolar interactions are the most efficient relaxation mechanism for these protons. However, the expected dependence of the relaxation rates as the sixth power of the distance has to be corrected because of induced relaxation among fast relaxing protons. This approach reveals that the solution structure of the protein is significantly different from the crystal structure around Phe-48. In addition, it provides an independent confirmation of the actual electronic structure of the [4Fe-4S]3+ cluster in the protein. The method devised in this work, which does not rely on specific enrichment, should be useful to improve the determination of NMR-derived solution structures of paramagnetic macromolecules.

Bacterial Proteins↗
PubMed1996

Nuclear-magnetic-resonance determination of the electron self-exchange rate constant of Clostridium pasteurianum rubredoxin.

The iron ion of rubredoxins efficiently exchanges one electron between the Fe(II) and Fe(III) oxidation states in mixtures of oxidized and reduced protein. The conditions under which the relaxation properties of the NMR signals can provide information about this exchange process have been worked out. The rate constant for the rubredoxin electron self-exchange ranges between 1.5 x 10(5) M-1 s-1 at 12 degrees C and 3 x 10(5) M-1 s-1 at 30 degrees C with an activation energy of the order of 24-30 kJ mol-1 in 50 mM potassium phosphate, pH 7. The increase of the electron self-exchange rate constant with ionic strength suggests that neutralizing electrostatic repulsion between the active sites of two molecules further accelerates the already fast electron exchange.

Bacterial Proteins↗
PubMed1996

The anaerobic Escherichia coli ribonucleotide reductase. Subunit structure and iron sulfur center.

During anaerobic growth Escherichia coli uses a specific ribonucleoside triphosphate reductase for the production of deoxyribonucleoside triphosphates. The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha 2) with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain. The radical is formed in an enzymatic reaction involving S-adenosylmethionine, NADPH, a reducing flavodoxin system and an additional 17.5-kDa polypeptide, previously called activase. Here, we demonstrate by EPR spectroscopy that this small protein contains a 4Fe-4S cluster that joins two peptides in a 35-kDa small homodimer (beta 2). A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase. These preparations were contaminated with a small amount of the small protein. The large and the small proteins form a tight complex. From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex. The anaerobic reductase thus has an alpha 2 beta 2 structure. We speculate that the small protein interacts with S-adenosylmethionine and forms a transient radical involved in the generation of the stable glycyl radical in the large protein that participates in the catalytic process.

Amino Acid Sequence↗
PubMed1996

Identification of residues of Rhodobacter capsulatus ferredoxin I important for its interaction with nitrogenase.

In Rhodobacter capsulatus, ferredoxin I (FdI) serves as natural electron donor to nitrogenase. In order to probe amino acid residues possibly involved in the interaction with dinitrogenase reductase, FdI was subjected to site-specific mutagenesis. A three-dimensional structure of FdI was designed by computer modelling and used for selecting target residues. Mutant ferredoxins bearing substitutions of surface residues, as well as a variant having a Met2 --> Tyr replacement in the vicinity of one cluster, have been constructed. All FdI variants were expressed to similar levels both in Escherichia coli and in a FdI-deleted mutant of the natural host. Once purified, the mutant ferredoxins exhibited molecular and spectroscopic properties almost identical to wild-type FdI. Determination of the reduction potential of FdI by cyclic voltammetry gave an E'o of -510 mV (pH 7.6) for both clusters, which is one of the lowest values reported for a 2[4Fe-4S] ferredoxin. Only the [Tyr2]FdI variant showed a significant difference in redox potential (delta E'o = -15 mV). Based on in vitro assays, a [Glu27, Glu28]FdI double mutant exhibited a twofold decrease in the electron transfer rate to dinitrogenase reductase while the affinity of this mutant for the enzyme was barely affected. On the other hand, an Asp36 --> His substitution resulted in a sevenfold increase of the apparent Km for dinitrogenase reductase. Unlike FdI and the other mutant ferredoxins, the [His36]FdI variant also failed to form a cross-linked complex with dinitrogenase reductase upon incubation with a carbodiimide. It is concluded that Asp36 in FdI probably participates in the interaction between the two protein partners. Nevertheless, all the FdI mutants proved competent in restoring a wild-type phenotype when expressed in a FdI-deleted mutant background, indicating that none of the studied residues was absolutely critical for electron transfer to nitrogenase.

Amino Acid Sequence↗
PubMed1996

Molecular mechanism of pyruvate-ferredoxin oxidoreductases based on data obtained with the Clostridium pasteurianum enzyme.

Pyruvate-ferredoxin oxidoreductase oxidises pyruvate in many fermentative microorganisms. The enzyme from Clostridium pasteurianum is an air-sensitive homodimer of 2x120000 daltons, for which pyruvate is the best substrate found among several alpha-ketoacids. Each subunit contains eight iron atoms in two [4Fe-4S] clusters. Two distinct EPR signals, possibly associated with two ligand environments, arise from one of these clusters. Binding of pyruvate does not generate a radical. The results reported suggest a scheme for the electron flow in pyruvate ferredoxin oxidoreductases according to which the detailed reaction mechanism depends on the number (even or odd) of [4Fe-4S] clusters present in a given enzyme.

Catalysis↗
PubMed1995

In vivo participation of a high potential iron-sulfur protein as electron donor to the photochemical reaction center of Rubrivivax gelatinosus.

We have found that the only high redox potential electron transfer component in the soluble fraction of Rubrivivax gelatinosus TG-9 is a high-potential iron-sulfur protein (HiPIP). We demonstrated the participation of this HiPIP in the photoinduced electron transfer both in vivo and in vitro. First, the addition of HiPIP to purified membranes enhanced the rate of re-reduction of the photooxidized reaction center. Second, the photooxidation of HiPIP was observed in intact cells of Ru. gelatinosus TG-9 under anaerobic conditions by EPR and absorption spectroscopies. Analysis of flash-induced absorption changes showed that the equilibration of positive equivalents between the reaction center and HiPIP occurs in less than 1 ms after flash excitation. The complete re-reduction of the photooxidized reaction center is achieved in tens of milliseconds. The turnover of a cyt bc1 is also involved in this reaction, as shown by a slow electrogenic phase of the membrane potential linked to this process.

Electron Spin Resonance Spectroscopy↗
PubMed1995

Characterization of a mutated rubredoxin with a cysteine ligand of the iron replaced by serine.

The active site of rubredoxins consists of a single iron tetrahedrally coordinated to four cysteinate sulfurs. One of the iron ligands, cysteine 42, has been mutated into serine in Clostridium pasteurianum rubredoxin. This mutation resulted in a shift to higher energy of the 320-800 nm region of the UV-visible absorption spectrum. Resonance Raman spectra showed that the nu 1 breathing mode of the iron chromophore was upshifted as a result of the C42S mutation. The spectral pattern, however, was not largely disturbed by the mutation. The EPR spectra of both the wild type and the C42S mutated protein displayed the characteristic features, at g = 4.3 and g = 9.5, of the "3/2" and "1/2" Kramers' doublets, respectively, of a S = 5/2 multiplet. These combined data afford strong evidence that in the C42S mutated rubredoxin serine has replaced cysteine 42 as a ligand of the iron, while maintaining the tetrahedral coordination of the metal. The most spectacular effect of the C42S mutation was a ca. 200 mV downshift of the redox potential of rubredoxin.

Base Sequence↗
PubMed1995

Characterization of a 2[4Fe-4S] ferredoxin obtained by chemical insertion of the Fe-S clusters into the apoferredoxin II from Rhodobacter capsulatus.

The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one [3Fe-4S] cluster and one [4Fe-4S] cluster. This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin. The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions. A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms. The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH. The reconstituted ferredoxin was purified and shown to be distinct from the native [7Fe-8S] ferredoxin, based on several biochemical and spectroscopic criteria. In the oxidized state, EPR revealed the quasi-absence of [3Fe-4S] cluster. 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2[4Fe-4S] ferredoxin. This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur. Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of [7Fe-8S] form. Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity. The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins.

Chromatography, Ion Exchange↗
PubMed1995

NMR of Chromatium vinosum ferredoxin: evidence for structural inequivalence and impeded electron transfer between the two [4Fe-4S] clusters.

The 2[4Fe-4S] ferredoxin from Chromatium vinosum has been investigated by 1H and 13C nuclear magnetic resonance. 1H NMR sequence-specific assignments have been obtained for a large majority of the residues. They indicate that the protein folds along a pattern similar to that previously evidenced for shorter 2[4Fe-4S] ferredoxins. However, C. vinosum ferredoxin differs from other ferredoxins by the occurrence of a turn in an eight amino acid region separating two successive cysteines, Cys-40 and Cys-49, liganding one cluster. Also, the unique C-terminal end of C. vinosum ferredoxin contains a 10 amino acid alpha-helix which interacts with one side of the above turn. The only cysteine of the sequence not involved in the ligation of the [4Fe-4S] clusters is Cys-57. Specific NMR experiments helped characterizing the signals arising from the ligands of these clusters: most of them display properties reminiscent of those of homologous ferredoxins, except for the signals associated with Cys-40. Despite the general similarity between C. vinosum ferredoxin and other 2[4Fe-4S] ferredoxins, the electron paramagnetic resonance and NMR spectra of the former reduced protein are significantly different from those previously observed for S = 1/2 [4Fe-4S]+ clusters. In addition, the intramolecular electron transfer rate in C. vinosum is far slower than in other similar cases. This is the first report of impeded electron exchange between two [4Fe-4S] clusters expected to be less than 12 A apart.

Amino Acid Sequence↗