The synthesis of acetyl-CoA by Clostridium thermoaceticum from carbon dioxide,hydrogen, coenzyme A and methyltetrahydrofolate

It has been demonstrated that enzymes from Clostridium thermoaceticum catalyze the following reaction in which Fd is ferredoxin and CH3THF is methyltetrahydrofolate. (for formula see text). The system involves hydrogenase, CO dehydrogenase, a methyltransferase, a corrinoid enzyme and other unknown components. Hydrogenase catalyzes the reduction of ferredoxin by H2; CO dehydrogenase then uses the reduced ferredoxin to reduce CO2 to a one-carbon intermediate that combines with CoASH and with a methyl group originating from CH3THF to form acetyl-CoA. It is proposed that these reactions are part of the mechanism which enables certain acetogenic autotrophic bacteria to grow on CO2 and H2.     

A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.     

Involvement of an activation protein in the methanol:2-mercaptoethanesulfonic acid methyltransferase reaction in Methanosarcina barkeri.

Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes required for transfer of the methyl group of methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 binds the methyl group of methanol to its corrinoid prosthetic group only when the central cobalt atom of the corrinoid is present in the highly reduced Co(I) state. However, upon manipulation of MT1 and even during catalysis, the enzyme becomes inactivated as the result of Co(I) oxidation. Reactivation requires H2, hydrogenase, and ATP. Ferredoxin stimulated the apparent reaction rate of methyl group transfer. Here we report that one more protein fraction was found essential for the overall reaction and, more specifically, for formation of the methylated MT1 intermediate. The more of the protein that was present, the shorter the delay of the start of methyl group transfer. The maximum velocity of methyl transfer was not substantially affected by these varying amounts of protein. This demonstrated that the protein was involved in the activation of MT1. Therefore, it was called methyltransferase activation protein.     

Purification and properties of ferredoxin and rubredoxin from Butyribacterium methylotrophicum.

A ferredoxin and a rubredoxin from Butyribacterium methylotrophicum, which displays a carbonyl-dependent acetyl-coenzyme A synthesis, were purified to electrophoretic homogeneity. The two electron carriers showed absorption spectra similar to those in Clostridium species. The ferredoxin displayed absorption peaks at 280 and 391 nm, while rubredoxin displayed absorption peaks at 279, 382, and 482 nm. Minimum molecular weights calculated from the respective amino acid compositions were 5,727 for ferredoxin and 5,488 for rubredoxin, excluding iron and inorganic sulfur atoms. Both electron carriers were isolated as monomers, according to gel-filtration data. Electron spin resonance analysis revealed that the ferredoxin was a 2[4Fe-4S]-type and that both clusters had a midpoint redox potential value of -410 mV, whereas rubredoxin contained one acid-stable iron and had a redox value of -40 mV. The coupling of these electron carriers to hydrogenase and carbon monoxide dehydrogenase activities was investigated. Rubredoxin showed higher activity towards carbon monoxide dehydrogenase, whereas ferredoxin showed higher activity towards hydrogenase.     

Purification and characterization of a heat stable ferredoxin isolated fromClostridium thermocellum

A thermostable ferredoxin was purified from Clostridium thermocellum. The final preparation was homogeneous as judged by electrophoresis in sodium dodecyl sulfate polyacrylamide gel and sedimentation equilibrium. It contains eight atoms of iron and eight acid-labile sulfur groups per molecule, the molecular weight is estimated to be 6 400 and the isoelectric point 3.35. Its amino-acid composition is characterized by the absence of histidine residues and the presence of eight cysteine residues. The absorption spectrum has a maximum at 390 nm with a molar absorption coefficient of 39 x 10(3) M1 cm-1, similar to that of other bacterial eight iron ferredoxins. The purified ferredoxin has high thermal stability, since the spectrophotometric absorption of the protein at 390 nm did not change after one hour at 70 degrees C and only thirty five per cent of absorbance were lost after one hour at 80 degrees C. With regard to the electron carrier activity, the stability is slightly higher, only twenty five per cent of the activity were lost after one hour at 80 degrees C. During pyruvate oxidation, ferredoxin functions in the transfer of electrons to hydrogenase and also in the back reaction during pyridine nucleotide reduction by a ferredoxin -NAD oxidoreductase using hydrogen as electron donor.     

Isolation, characterization, and biological activity of the Methanococcus thermolithotrophicus ferredoxin.

A ferredoxin has been isolated from the thermophilic methanogen Methanococcus thermolithotrophicus. The native protein was a monomer exhibiting a molecular weight of 7,262, calculated from the amino acid composition. Its absorption spectrum had two maxima at 390 and 283 nm, with an absorbance ratio A390/A283 of 0.79. The absorption at 390 nm (E = 29 mM-1 cm-1) and the content of iron of the protein are in agreement with the presence of two 4Fe-4S clusters in M. thermolithotrophicus ferredoxin. Its amino acid composition showed the presence of eight cysteine residues, which is the required number of cysteines for the binding of two 4Fe-4S clusters. The protein was characterized by the lack of histidine, arginine, and leucine and a high content of valine. It was unusually stable to high temperatures but not to oxygen. The ESR spectrum of the protein in the oxidized state showed a minor signal at g = 2.01, corresponding to an oxidized 3Fe-4S cluster. The protein, which was difficult to reduce with dithionite or reduced mediators, exhibited in its reduced state a spectrum typical of two interacting reduced 4Fe-4S clusters. M. thermolithotrophicus ferredoxin functioned as an electron acceptor for the CO dehydrogenase complex with an extract free of ferredoxin. No reaction was detected with F420 or hydrogenase.     

Purification and properties of methanol:5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarcina barkeri

Methanol:5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarcina barkeri has been purified to approximately 90% homogeneity by ion-exchange chromatography on DEAE-cellulose and QAE-A50 Sephadex columns. The molecular weight, estimated by gel electrophoresis, was found to be 122,000, and the enzyme contained two different subunits with molecular weights of 34,000 and 53,000, which indicates an alpha 2 beta structure. The enzyme contains three or four molecules of 5-hydroxybenzimidazolylcobamide, which could be removed by treatment of the enzyme with 2-mercaptoethanol or sodium dodecyl sulfate. In both cases the enzyme dissociated into its subunits. For stability, the enzyme required the presence of divalent cations such as Mg2+, Mn2+, Sr2+, Ca2+, or Ba2+. ATP, GTP, or CTP was needed in a reductive activation process of the enzyme. This activation was brought about by a mixture of H2, ferredoxin, and hydrogenase, but also by CO, which is thought to reduce the corrinoid chemically. The CO dehydrogenase-like activity of the methyltransferase is discussed.     

On the reduction of aliphatic and aromatic nitro compounds by Clostridia, the role of ferredoxin and its stabilization

Crude extracts of a variety of Clostridium species reduce aromatic and aliphatic nitro compounds in the presence of hydrogen gas. Using different Clostridia, the uptake of hydrogen by p-nitrobenzoate is about 5--10 times faster than by 2-nitroethanol. Structurally rather different aliphatic nitro compounds show rates which differ by less than a factor of 3. Hydrogenase from Clostridium kluyveri and ferredoxins from Clostridium spec. La 1 and spinach have been purified. The combinations of the hydrogenase and each one of the ferredoxins catalyse the hydrogen uptake by nitro compounds. Clostridial flavodoxin also transfer electrons onto nitro compounds. Nitroaryl and nitroalkyl compounds behave differently with ferredoxin. The first reduction step (1-electron transfer) of p-nitrobenzoate leads to the nitro radical anion which can be detected by EPR measurements. Nitro alkanes seem to form a rather unstable radical which decomposes partially to form nitrite. Furthermore, 2-(N-hydroxyimino)- and 2-(N-hydroxyamino)ethanol, a nitrogen radical of 2-(N-hydroxyamino)ethanol as well as glycol and 1,4-butanediol were detected as intermediates and side products during the reduction of 2-nitro-ethanol to 2-aminoethanol. While the hydrogenase from Clostridium kluyveri seems not to be affected by any reduction intermediate, the ferredoxin from Clostridium spec. La 1 is inactivated by nitrite in a few minutes. Ferrous and sulfide ions in concentrations substoichiometric to that of nitrite stabilize and even reactivate the ferredoxin in the presence of 2-mercaptoethanol. A mechanism for the reduction of aliphatic nitro compounds catalysed by hydrogenase and ferredoxin is proposed.     

Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri.

Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U x mg protein-1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 microM. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U x mg protein-1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 microM. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 microM. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.     

MT FdR: a ferredoxin reductase from M. tuberculosis that couples to MT CYP51

We report the molecular cloning, expression and partial characterization of MT FdR, an FAD-associated flavoprotein, from Mycobacterium tuberculosis similar to the oxygenase-coupled NADH-dependent ferredoxin reductases (ONFR). We establish, through kinetic and spectral analysis, that MT FdR preferentially uses NADH as cofactor. Furthermore, MT FdR forms a complex with mycobacterial ferredoxin (MT Fdx) and MT CYP51, a cytochrome P450 (CYP) from M. tuberculosis that is similar to lanosterol 14alpha-demethylase isozymes. This reconstituted system transfers electrons from the cofactor to the heme iron of MT CYP51 and effects the demethylation of lanosterol.     

Studies on nitrate reductase of Clostridium perfringens. II. Purification and some properties of ferredoxin.

A ferredoxin was purified from Clostridium perfringens by DEAE-cellulose chromatography and Sephadex G-50 gel filtration. It had absorption maxima at 390 and 280 nm. The molecular weight was estimated to be 6,000 by Sephadex gel filtration and from the results of amino acid analysis. The isoelectric point was 3.0. It contained four atoms of iron, four atoms of labile sulfur, and six cysteine residues. This ferredoxin as well as ferredoxin from C. pasteurianum acted as an electron donor for nitrate reductase from C. perfringens. The ferredoxin could also act as an electron donor for the hydrogenase from C. pasteurianum in hydrogen evolution.     

Hydrogenosomes in the rumen fungus Neocallimastix patriciarum.

Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.     

Efficiency of ferredoxins and flavodoxins as mediators in systems for hydrogen evolution.

1. The efficiencies of ferredoxins and flavodoxins from a range of sources as mediators in systems for hydrogen evolution were assessed. 2. In supporting electron transfer from dithionite to hydrogenase of the bacterium Clostridium pasteurianum, highest activity was shown by the ferredoxin from the cyanobacterium Chlorogloeopsis fritschii and flavodoxin from the bacterium Megasphaera elsdenii. The latter was some twenty times as active as comparable concentrations of Methyl Viologen. Ferredoxins from the cyanobacterium Anacystis nidulans and the red alga Porphyra umbilicalis also showed high activity. 3. In mediating electron transfer from chloroplast membranes to Clostridium pasteurianum hydrogenase the flavodoxin from Anacystis nidulans proved the most active with Nostoc strain MAC flavodoxin and Porphyra umbilicalis ferredoxin also being appreciably more active than other cyanobacterial and higher plant ferredoxins. 4. In both hydrogenase systems the ferredoxin and flavodoxin from the red alga Chondrus crispus and the ferredoxin from another red alga Gigartina stellata showed very low activity. 5. There appeared to be no apparent correlation of efficiency in supporting hydrogenase activity with midpoint redox potential (Em) of the mediators, though some correlation of Em with the efficiency of the mediators in supporting NADP+ photoreduction by chloroplasts, or pyruvate oxidation by a Clostridium pasteurianum system, was evident. 6. Activity of the mediators in the hydrogenase systems therefore primarily reflects differences in tertiary structure conferring differing affinities for the other components of the systems.     

M?ssbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl coenzyme A by Clostridium thermoaceticum

We have purified to homogeneity the 88-kDa corrinoid protein from Clostridium thermoaceticum which acts as a methyl carrier in the synthesis of acetyl-CoA. As shown here, this protein contains a [4Fe-4S]1+/2+ cluster in addition to a corrinoid. The corrinoid is 5-methoxybenzimidazolylcobamide, with an OH- group probably present as the upper axial ligand. Co+ is present in the reduced form, Co2+ in the as-isolated form, and Co3+ in the methylated form of the protein. The as-isolated corrinoid/Fe-S protein exhibits a Co2+ EPR signal lacking nitrogen superhyperfine splittings, indicating that the benzimidazole base is uncoordinated ("base-off") in the Co2+ state. Optical studies suggest that the Co3+-CH3 corrinoid is also base-off. In the as-isolated and methylated forms, the iron-sulfur cluster is diamagnetic, with quadrupole splittings and isomer shifts characteristic of [4Fe-4S]2+ clusters. The protein can be reduced by CO and CO dehydrogenase in the absence of ferredoxin. The EPR spectra of the reduced cluster exhibit two components: one with principal g-values at 2.07, 1.93, and 1.82 and the other at 2.02, 1.94, and 1.86. The M?ssbauer data show that these signals result from [4Fe-4S]1+ clusters. Chemical analysis shows that the iron:cobalt atomic ratio is close to 4:1, suggesting that a single [4Fe-4S]1+ cluster occurs in two distinct S = 1/2 spin states in the reduced state. Treatment with 1-2.5 M urea converts the two cluster forms into a single one, with EPR and M?ssbauer spectra of typical [4Fe-4S]1+ clusters. A 27-kDa corrinoid protein (Ljungdahl, L.G., LeGall, J., and Lee, J.P. (1973) Biochemistry 12, 1802-1808) also was purified and found to be inactive in the synthesis of acetyl-CoA, contrary to the suggestion of Ljungdahl et al. (1973).     

Brownian dynamics and molecular dynamics study of the association between hydrogenase and ferredoxin from Chlamydomonas reinhardtii

The [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii can catalyze the reduction of protons to hydrogen gas using electrons supplied from photosystem I and transferred via ferredoxin. To better understand the association of the hydrogenase and the ferredoxin, we have simulated the process over multiple timescales. A Brownian dynamics simulation method gave an initial thorough sampling of the rigid-body translational and rotational phase spaces, and the resulting trajectories were used to compute the occupancy and free-energy landscapes. Several important hydrogenase-ferredoxin encounter complexes were identified from this analysis, which were then individually simulated using atomistic molecular dynamics to provide more details of the hydrogenase and ferredoxin interaction. The ferredoxin appeared to form reasonable complexes with the hydrogenase in multiple orientations, some of which were good candidates for inclusion in a transition state ensemble of configurations for electron transfer.     

A Bacterial Electron-bifurcating Hydrogenase

The Wood-Ljungdahl pathway of anaerobic CO(2) fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD(+) for this reaction. NAD(+) was also reduced but only in the presence of ferredoxin. NAD(+) and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD(+) and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.     

Effects of electron mediator charge properties on the reaction kinetics of hydrogenase from Chlamydomonas

Anions modulate hydrogenase activity in cell-free preparations of Chlamydomonas reinhardtii, and this modulation is greatly influenced by the charge properties of the redox agent included to mediate electron transfer to hydrogenase. With cationic methyl viologen as the electron mediator, anions stimulate the maximum velocity of H2 production (e.g., a 320% increase in the presence of 1 M NaCl) but have little effect on the Km for methyl viologen. Conversely, when hydrogenase activity is mediated by polyanionic metatungstate or ferredoxin, H2 production is strongly inhibited by anions (e.g., 70-77% inhibition by 0.2 M NaCl). This inhibition is primarily due to a reduced affinity of hydrogenase for these mediators (as evidenced by a large increase in Km values), rather than a change in the maximum velocity of the reaction. Anions have little effect on the kinetics of hydrogenase activity mediated by zwitterionic sulfonatopropyl viologen, a redox agent with a nearly neutral net charge. These results suggest the presence of a cationic region near the active site of hydrogenase. This cationic region, probably due to lysine and/or arginine residues, may serve in vivo to facilitate the interaction between hydrogenase and ferredoxin, the polyanionic, physiological electron mediator.     

NADH:ferredoxin reductase and NAD-reducing hydrogenase activities in Hydrogenobacter thermophilus strain TK-6

Abstract NADH:ferredoxin reductase (EC 1.18.1.3) and NAD-reducing hydrogenase (EC 1.12.1.2) activities were detected in the cytoplasm of Hydrogenobacter thermophilus TK-6. NADH:ferredoxin reductase activity was detected using metronidazole, an artificial electron acceptor, which reacts specifically with reduced ferredoxin. Soluble NAD-reducing hydrogenase activity was detected after extended preincubation. The lag disappeared when cell-free extract was incubated anaerobically for more than 30 min. The electron transport system of this chemolithoautotrophic bacterium is discussed.     

Purification and properties of hydrogenase from Megasphaera elsdenii

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacterium Megasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain with Mr approximately 50 000 which contains 12 atoms of non-haem iron and 12 atoms of acid-labile sulphide. The enzyme is rapidly inactivated by O2 and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (epsilon 275 nm = 143 mM-1 cm-1) and a shoulder between 350 nm and 400 nm (epsilon 400 nm = 46 mM-1 cm-1). The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co-operativity, but no indication of self-association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin. Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co-operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one-electron donors flavodoxin and methyl viologen semiquinone monomer and the two-electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe-4S) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer. The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis-Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two-electron acceptors used are consistent with the porposed models, if it is assumed that the pathway followed is compulsory. At this moment no choice can be made between the models proposed.     

NMR studies of electron carrier proteins from sulphate reducing bacteria.

The sulphate-reducing bacteria have a complex electron transfer system which leads to the reduction of sulphate by oxidation of either organic substrates or molecular hydrogen. These bacteria can either produce or consume molecular hydrogen. The central part of this electron pathway for Desulovibrio gigas is constituted by hydrogenase (3 X (4Fe-4S)). cytochrome c3 (4 haems with different redox potentials) and a one (4Fe-4S) cluster ferredoxin. This ferredoxin is isolated in different oligomeric forms, which stabilize different oxidation states and have different physiological roles; the trimer FdI being involved in the production of H2 and the tetramer FdII being more efficient for the consumption of H2. The presence of intrinsic probes (the iron ions) in these proteins is particularly helpful for structural studies using NMR spectroscopy. These studies allowed a characterization of the oxidation states used by the different oligomers of the ferredoxin and obtaintion of structural information on multi-haem cytochromes (c3 and c7). NMR is also suitable to study protein-protein interaction. The study of the complex formed between FdII and cytochrome c3 has shown that there is an alteration of the kinetics of electron transfer upon complexation.