Kinetics for formate dehydrogenase of Escherichia coli formate-hydrogenlyase

Kinetic parameters of the selenium-containing, formate dehydrogenase component of the Escherichia coli formate-hydrogenlyase complex have been determined with purified enzyme. A ping-pong Bi Bi kinetic mechanism was observed. The Km for formate is 26 mM, and the Km for the electron-accepting dye, benzyl viologen, is in the range 1-5 mM. The maximal turnover rate for the formate-dependent catalysis of benzyl viologen reduction was calculated to be 1.7 x 10(5) min-1. Isotope exchange analysis showed that the enzyme catalyzes carbon exchange between carbon dioxide and formate in the absence of other electron acceptors, confirming the ping-pong reaction mechanism. Dissociation constants for formate (12.2 mM) and CO2 (8.3 mM) were derived from analysis of the isotope exchange data. The enzyme catalyzes oxidation of the alternative substrate deuterioformate with little change in the Vmax, but the Km for deuterioformate is approximately three times that of protioformate. This implies formate oxidation is not rate-limiting in the overall coupled reaction of formate oxidation and benzyl viologen reduction. The deuterium isotope effect on Vmax/Km was observed to be approximately 4.2-4.5. Sodium nitrate was found to inhibit enzyme activity in a competitive manner with respect to formate, with a Ki of 7.1 mM. Sodium azide is a noncompetitive inhibitor with a Ki of about 80 microM.     

Periplasmic methacrylate reductase activity in Wolinella succinogenes

The cell homogenate and the soluble cell fraction of Wolinella succinogenes grown with formate and fumarate catalyzed the oxidation of benzyl viologen radical by methacrylate [apparent Km=0.23 mM, Vmax=1.0 U (mg cell protein) -1] or acrylate [apparent Km=0.50 mM, Vmax=0.77 U (mg cell protein) -1]. Crotonate did not serve as an oxidant. A mutant of W. succinogenes lacking the fccABC operon was unable to catalyze methacrylate or acrylate reduction. In contrast, the inactivation of fccC alone had no effect on these activities. Methacrylate reduction by benzyl viologen radical was not catalyzed by fumarate reductase isolated from the membrane of W. succinogenes. Cells grown with formate and fumarate did not catalyze methacrylate reduction by formate, and W. succinogenes did not grow with formate and methacrylate as catabolic substrates. The results suggest that the reduction of methacrylate or acrylate by benzyl viologen radical is most likely catalyzed either by the periplasmic flavoprotein FccA or by a complex consisting of FccA and the predicted c-type cytochrome FccB. The metabolic function of the fccABC operon remains unknown.     

Conversion of the central [4Fe-4S] cluster into a [3Fe-4S] cluster leads to reduced hydrogen-uptake activity of the F420-reducing hydrogenase of Methanococcus voltae.

As in many other hydrogenases, the small subunit of the F420-reducing hydrogenase of Methanococcus voltae contains three iron-sulfur clusters. The arrangement of the three [4Fe-4S] clusters corresponds to the arrangement of [Fe-S] clusters in the [NiFeSe] hydrogenase of Desulfomicrobium baculatum. Many other hydrogenases contain two [4Fe-4S] clusters and one [3Fe-4S] cluster with a relatively high redox potential, which is located in the central position between a proximal and a distal [4Fe-4S] cluster. We have investigated the role of the central [4Fe-4S] cluster in M. voltae with regard to its effect on the enzyme activity and its spectroscopic properties. Using site-directed mutagenesis, we constructed a strain in which one cysteine ligand of the central [4Fe-4S] cluster was replaced by proline. The mutant protein was purified, and the [4Fe-4S] to [3Fe-4S] cluster conversion was confirmed by EPR spectroscopy. The conversion resulted in an increase in the redox potential of the [3Fe-4S] cluster by about 400 mV. The [NiFe] active site was not affected significantly by the mutation as assessed by the unchanged Ni EPR spectrum. The specific activity of the mutated enzyme did not show any significant differences with the artificial electron acceptor benzyl viologen, but its specific activity with the natural electron acceptor F420 decreased tenfold.     

Tricarballylate catabolism in Salmonella enterica. The TcuB protein uses 4Fe-4S clusters and heme to transfer electrons from FADH2 in the tricarballylate dehydrogenase (TcuA) enzyme to electron acceptors in the cell membrane

Tricarballylate, a citrate analogue, is considered the causative agent of grass tetany, a ruminant disease characterized by acute magnesium deficiency. Although the normal rumen flora cannot catabolize tricarballylate, the Gram-negative enterobacterium Salmonella enterica can. An operon dedicated to tricarballylate utilization (tcuABC) present in this organism encodes all functions required for tricarballylate catabolism. Tricarballylate is converted to the cis-aconitate in a single oxidative step catalyzed by the FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme. We hypothesized that the uncharacterized TcuB protein was required to reoxidize the flavin cofactor in vivo. Here, we report the initial biochemical characterization of TcuB. TcuB is associated with the cell membrane and contains two 4Fe-4S clusters and heme. Site-directed mutagenesis of cysteinyl residues putatively required as ligands of the 4Fe-4S clusters completely inactivated TcuB function. TcuB greatly increased the Vmax of the TcuA reaction from 69 +/- 2 to 8200 +/- 470 nmol min-1 mg-1; the Km of TcuA for tricarballylate was unaffected. Inhibition of TcuB activity by an inhibitor of ubiquinone oxidation, 2,5-dibromo-3-methyl-6-isoproylbenzoquinone (DBMIB), implicated the quinone pool as the ultimate acceptor of electrons from FADH2. We propose a model for the electron flow from FADH2, to the 4Fe-4S clusters, to the heme, and finally to the quinone pool.     

Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Electron-paramagnetic-resonance studies on the molybdenum centre.

Formate dehydrogenase from Pseudomonas aeruginosa contains molybdenum, a [4Fe-4S] cluster and cytochrome b. This paper reports the detection of molybdenum as Mo(V) by e.p.r. spectroscopy. In order to generate Mo(V) signals, addition of amounts of excess formate varying between 10- and 50-fold over enzyme, followed by 200-fold excess of sodium dithionite, were used. Two Mo(V) species were observed. One, the major component, has g1 = 2.012, g2 = 1.985 and g3 = 1.968, appeared at low concentrations of formate and increased linearly in intensity with increasing concentrations of formate up to 25-fold excess over the enzyme. At higher formate concentration this signal disappeared. The appearance and disappearance of this Mo(V) signal seems to parallel the state of reduction of the [4Fe-4S] clusters. A second, minor, Mo(V) species with g-values g1 = 1.996, g2 = 1.981 and g3 = 1.941 appears at a constant level during the formate-dithionite titration. No evidence has been obtained for nuclear hyperfine coupling to protons. The major Mo(V) species has unusual e.p.r. signals compared with other molybdenum-containing enzymes, except for that observed in the formate dehydrogenase from Methanobacterium formicicum [Barber, Siegel, Schauer, May & Ferry (1983) J. Biol. Chem. 258, 10839-10845]. The present work suggests that the enzyme is acting as a CO2 reductase, with dithionite as an electron donor to a [4Fe-4S] cluster, which in turn donates electrons to molybdenum, producing a Mo(V) species with CO2 bound to the metal.     

Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 +/- 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (kcat=37.8 +/- 3 s(-1); Km=30 +/- 3 mM) and 2-oxobutyrate (kcat=33.2 +/- 3 s(-1); Km=90 +/- 8 mM). Thiamine pyrophosphate (Km=1 microM) and certain divalent metal ions such as Mg2+ (Km=29 microM), Mn2+ (Km=2 microM), and Co2+ (Km=11 microM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km=106 microM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.     

Formate dehydrogenase activity in cells and outer membrane blebs of Desulfovibrio gigas

Formate dehydrogenase in Desulfovibrio gigas was measured by following the release of 14CO2 from radiolabeled formate. Experiments with whole cells using sulfate as the electron acceptor revealed optimal formate dehydrogenase activity at pH 7.0 and formate utilization followed saturation kinetics. While formate dehydrogenase was constitutively produced in pyruvate or lactate media, the formate dehydrogenase activity was markedly increased in cells grown with formate as the electron donor. In cell-free experiments with methyl viologen or 2,6 dichlorophenolindophenol, about 1% of the cellular formate dehydrogenase activity was present in blebs from the outer membrane. Electron microscopy revealed that these blebs were closed structures with diameters ranging from 80-800A and were not induced by changes in osmotic pressure or cellular autolysis. Analysis of blebs revealed the presence of lipopolysaccharides and two proteins with molecular masses of 70 and 53 kDa.     

Oxalate metabolism by the acetogenic bacterium Moorella thermoacetica

Whole-cell and cell-extract experiments were performed to study the mechanism of oxalate metabolism in the acetogenic bacterium Moorella thermoacetica. In short-term, whole-cell assays, oxalate consumption was low unless cell suspensions were supplemented with CO(2), KNO(3), or Na(2)S(2)O(3). Cell extracts catalyzed the oxalate-dependent reduction of benzyl viologen. Oxalate consumption occurred concomitant to benzyl viologen reduction; when benzyl viologen was omitted, oxalate was not appreciably consumed. Based on benzyl viologen reduction, specific activities of extracts averaged 0.6 micromol oxalate oxidized min(-1) mg protein(-1). Extracts also catalyzed the formate-dependent reduction of NADP(+); however, oxalate-dependent reduction of NADP(+) was negligible. Oxalate- or formate-dependent reduction of NAD(+) was not observed. Addition of coenzyme A (CoA), acetyl-CoA, or succinyl-CoA to the assay had a minimal effect on the oxalate-dependent reduction of benzyl viologen. These results suggest that oxalate metabolism by M. thermoacetica requires a utilizable electron acceptor and that CoA-level intermediates are not involved.     

Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Uptake hydrogenase (EC 1.12) from Azotobacter vinelandii has been purified 250-fold from membrane preparations. Purification involved selective solubilization of the enzyme from the membranes, followed by successive chromatography on DEAE-cellulose, Sephadex G-100, and hydroxylapatite. Freshly isolated hydrogenase showed a specific activity of 110 mumol of H2 uptake (min X mg of protein)-1. The purified hydrogenase still contained two minor contaminants that ran near the front on sodium dodecyl sulfate-polyacrylamide gels. The enzyme appears to be a monomer of molecular weight near 60,000 +/- 3,000. The pI of the protein is 5.8 +/- 0.2. With methylene blue or ferricyanide as the electron acceptor (dyes such as methyl or benzyl viologen with negative midpoint potentials did not function), the enzyme had pH optima at pH 9.0 or 6.0, respectively, It has a temperature optimum at 65 to 70 degrees C, and the measured half-life for irreversible inactivation at 22 degrees C by 20% O2 was 20 min. The enzyme oxidizes H2 in the presence of an electron acceptor and also catalyzes the evolution of H2 from reduced methyl viologen; at the optimal pH of 3.5, 3.4 mumol of H2 was evolved (min X mg of protein)-1. The uptake hydrogenase catalyzes a slow deuterium-water exchange in the absence of an electron acceptor, and the highest rate was observed at pH 6.0. The Km values varied widely for different electron acceptors, whereas the Km for H2 remained virtually constant near 1 to 2 microM, independent of the electron acceptors.     

Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans

Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans. Both enzymes differ in their subunit molecular masses, N-terminal sequences, peptide maps, and immunological reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in association with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO + H(2)O --> CO(2) + 2 e(-) + 2 H(+). Oxidized viologen dyes are effective electron acceptors. The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) micromol of CO oxidized min(-1) mg(-1) of protein (methyl viologen, pH 8.0, 70 degrees C). The two enzymes oxidize CO very efficiently, as indicated by k(cat)/K(m) values at 70 degrees C of 1.3. 10(9) M(-1) CO s(-1) (CODH I) and 1.7. 10(9) M(-1) CO s(-1) (CODH II). The apparent K(m) values at pH 8.0 and 70 degrees C are 30 and 18 microM CO for CODH I and CODH II, respectively. Acetyl coenzyme A synthase activity is not associated with the enzymes. CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers containing 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, respectively. Electron paramagnetic resonance (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.     

The membrane-bound hydrogenase from Paracoccus denitrificans. Purification and molecular characterization

The membrane-bound hydrogenase from Paracoccus denitrificans was purified 68-fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 mumol H2 min-1 (mg protein)-1 (methylene blue reduction). Purification involved solubilization by Triton X-114, phase separation, chromatography on DEAE-Sephacel, ammonium-sulfate precipitation and chromatography on Procion-red HE-3B-Sepharose. Gel electrophoresis under denaturing conditions revealed two non-identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100 kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane-bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half-life for inactivation under air at 20 degrees C was 8 h. The Paracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c, FMN, 2,6-dichloroindophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue (V = 161.9 U/mg; Km = 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively). The native hydrogenase from P. denitrificans cross-reacted with purified antibodies raised against the membrane-bound hydrogenase from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.     

Purification, characterization, and metabolic function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon Thermococcus strain ES-1.

Thermococcus strain ES-1 is a strictly anaerobic, hyperthermophilic archaeon that grows at temperatures up to 91 degrees C by the fermentation of peptides. It is obligately dependent upon elemental sulfur (S(o)) for growth, which it reduces to H2S. Cell extracts contain high aldehyde oxidation activity with viologen dyes as electron acceptors. The enzyme responsible, which we term aldehyde ferredoxin oxidoreductase (AOR), has been purified to electrophoretic homogeneity. AOR is a homodimeric protein with a subunit M(r) of approximately 67,000. It contains molybdopterin and one W, four to five Fe, one Mg, and two P atoms per subunit. Electron paramagnetic resonance analyses of the reduced enzyme indicated the presence of a single [4Fe-4S]+ cluster with an S = 3/2 ground state. While AOR oxidized a wide range of aliphatic and aromatic aldehydes, those with the highest apparent kcat/Km values (> 10 microM-1S-1) were acetaldehyde, isovalerylaldehyde, and phenylacetaldehyde (Km values of < 100 microM). The apparent Km value for Thermococcus strain ES-1 ferredoxin was 10 microM (with crotonaldehyde as the substrate). Thermococcus strain ES-1 AOR also catalyzed the reduction of acetate (apparent Km of 1.8 mM) below pH 6.0 (with reduced methyl viologen as the electron donor) but at much less than 1% of the rate of the oxidative reaction (with benzyl viologen as the electron acceptor at pH 6.0 to 10.0). The properties of Thermococcus strain ES-1 AOR are very similar to those of AOR previously purified from the saccharolytic hyperthermophile Pyrococcus furiosus, in which AOR was proposed to oxidize glyceraldehyde as part of a novel glycolytic pathway (S. Mukund and M. W. W. Adams, J. Biol. Chem. 266:14208-14216, 1991). However, Thermococcus strain ES-1 is not known to metabolize carbohydrates, and glyceraldehyde was a very poor substrate (kcat/Km of < 0.2 microM-1S-1) for its AOR. The most efficient substrates for Thermococcus strain ES-1 AOR were the aldehyde derivatives of transaminated amino acids. This suggests that the enzyme functions to oxidize aldehydes generated during amino acid catabolism, although the possibility that AOR generates aldehydes from organic acids produced by fermentation cannot be ruled out.     

Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum

Methylenetetrahydrofolate reductase in Clostridium formicoaceticum has been purified to a specific activity of 140 mumol min-1 mg-1 when assayed at 37 degrees C, pH 7.2, in the direction of oxidation of 5-methyltetrahydrofolate with benzyl viologen as electron acceptor. The purified enzyme is judged to be homogeneous by polyacrylamide disc-gel electrophoresis and gel filtration. The enzyme which is an octamer has a molecular weight of about 237,000 and consists of four each of two different subunits having the molecular weights 26,000 and 35,000. The octameric enzyme contains per mol 15.2 +/- 0.3 iron, 2.3 +/- 0.2 zinc, 19.5 +/- 1.3 acid-labile sulfur, and 1.7 FAD. The UV-visible absorbance spectrum has a peak at 385 nm and a shoulder at 430 nm and is that of a flavoprotein containing iron-sulfur centers. The reductase, which is sensitive to oxygen, must be handled anaerobically and is stabilized by 2 mM dithionite. It catalyzes the reduction of methylene blue, menadione, benzyl viologen, rubredoxin, and FAD with 5-methyltetrahydrofolate and the oxidation of reduced ferredoxin and FADH2 with 5,10-methylenetetrahydrofolate. No activity was observed with pyridine nucleotides. It is suggested that the physiologically important reaction catalyzed by the enzyme is the reduced ferredoxin-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

Purifications and characterizations of a ferredoxin and its related 2-oxoacid:ferredoxin oxidoreductase from the hyperthermophilic archaeon, Sulfolobus solfataricus P1

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of 11,180+/-50 Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa alpha and 34 kDa beta subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at 55 degrees C, pH 7.0. Maximum activity was observed at 70 degrees C and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with Km and kcat values of 163 microM and 452 min(-1), respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.     

Studies on the Induction of Reversible Hydrogenase from Cyanobacterium Anabaena variabilis and its Properties

The reversible hydrogenase in vegetative cells of A. variabilis cultured on NH4+ or N-free medium was induced by sparging with N2 for 24 hours under light. Both anaerobic condition and illumination appear to be necessary for the induction of hydrogenase in this algae. The properties of the hydrogenase in cell-free extract obtained from the cells grown on two nitrogen sources are similar: (1) Both the enzymes are able to evolve H2 in the presence of reduced methyl viotogen as electron donor, and to uptake H2 in the presence of benzyl viologen as electron acceptor. (2) The enzymes posses the thermal stability and are stable to O2. (3) The optimum pH required for H2 evolution activity of the enzymes is 7.0–7 5. (4) The Km of the enzymes obtained from NH4+ grown cells and N-free grown cells is 300 mmol/l and 295 mmol/l, respectively. So the high Km measured here suggests that the enzymes in both cases function physiologically as H2 evolution. (5) The activities of both enzymes are inhibited by CO but are not affected by C2H2. The induced H2 evolution activity of the reversible hydrogenase in cells grown on NH4+ reached 1530 nmol H2/mg dry wt, h, which was 3 to 5 times higher than from cells grown on N-free medium. Our experiment results indicate that the appearance of heterocysts of A. variabilis cultured on N-free medium affects the synthesis of reversible hydrogenase and the regulation of its activity.     

Diphenylene iodonium as an inhibitor for the hydrogenase complex of Rhodobacter capsulatus. Evidence for two distinct electron donor sites

The photosynthetic bacterium Rhodobacter capsulatus synthesises a membrane-bound [NiFe] hydrogenase encoded by the H2 uptake hydrogenase (hup)SLC structural operon. The hupS and hupL genes encode the small and large subunits of hydrogenase, respectively; hupC encodes a membrane electron carrier protein which may be considered as the third subunit of the uptake hydrogenase. In Wolinella succinogenes, the hydC gene, homologous to hupC, has been shown to encode a low potential cytochrome b which mediates electron transfer from H2 to the quinone pool of the bacterial membrane. In whole cells of R. capsulatus or intact membrane preparation of the wild type strain B10, methylene blue but not benzyl viologen can be used as acceptor of the electrons donated by H2 to hydrogenase; on the other hand, membranes of B10 treated with Triton X-100 or whole cells of a HupC- mutant exhibit both benzyl viologen and methylene blue reductase activities. We report the effect of diphenylene iodonium (Ph2I), a known inhibitor of mitochondrial complex I and of various monooxygenases on R. capsulatus hydrogenase activity. With H2 as electron donor, Ph2I inhibited partially the methylene blue reductase activity in an uncompetitive manner, and totally benzyl viologen reductase activity in a competitive manner. Furthermore, with benzyl viologen as electron acceptor, Ph2I increased dramatically the observed lagtime for dye reduction. These results suggest that two different sites exist on the electron donor side of the membrane-bound [NiFe] hydrogenase of R. capsulatus, both located on the small subunit. A low redox potential site which reduces benzyl viologen, binds Ph2I and could be located on the distal [Fe4S4] cluster. A higher redox potential site which can reduce methylene blue in vitro could be connected to the high potential [Fe3S4] cluster and freely accessible from the periplasm.     

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.     

Characterization of the iron-sulfur clusters in ferredoxin from acetate-grown Methanosarcina thermophila.

Ferredoxin from Methanosarcina thermophila is an electron acceptor for the CO dehydrogenase complex which decarbonylates acetyl-coenzyme A and oxidizes the carbonyl group to carbon dioxide in the pathway for conversion of the methyl group of acetate to methane (K. C. Terlesky and J. G. Ferry, J. Biol. Chem. 263:4080-4082, 1988). Resonance Raman spectroscopy and electron paramagnetic resonance spectroelectrochemistry indicated that the ferredoxin contained two [4Fe-4S] clusters per monomer of 6,790 Da, each with a midpoint potential of -407 mV. A [3Fe-4S] species, with a midpoint potential of +103 mV, was also detected in the protein at high redox potentials. Quantitation of the [3Fe-4S] and [4Fe-4S] centers revealed 0.4 and 2.1 spins per monomer, respectively. The iron-sulfur clusters were unstable in the presence of air, and the rate of cluster loss increased with increasing temperature. A ferredoxin preparation, with a low spin quantitation of [4Fe-4S] centers, was treated with Fe2+ and S2-, which resulted in an increase in [4Fe-4S] and a decrease in [3Fe-4S] clusters. The results of these studies suggest the [3Fe-4S] species may be an artifact formed from degradation of [4Fe-4S] clusters.     

Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101.

Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor. DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity). A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity. Methyl viologen, reduced flavin mononucleotide, and reduced flavin adenine dinucleotide also served as electron donors for DMSO reduction. Methionine sulfoxide, a DMSO analog, could substitute for DMSO in both the growth medium and in the benzyl viologen assay. DMSO reductase activity was present in cells grown anaerobically on DMSO but was repressed by the presence of nitrate or by aerobic growth. Anaerobic growth on DMSO coinduced nitrate, fumarate, and and trimethylamine-N-oxide reductase activities. The requirement of a molybdenum cofactor for DMSO reduction was suggested by the inhibition of growth and a 60% reduction in DMSO reductase activity in the presence of 10 mM sodium tungstate. Furthermore, chlorate-resistant mutants chlA, chlB, chlE, and chlG were unable to grow anaerobically on DMSO. DMSO reduction appears to be under the control of the fnr gene.