Electron bifurcation involved in the energy metabolism of the acetogenic bacterium Moorella thermoacetica growing on glucose or H2 plus CO2

Moorella thermoacetica ferments glucose to three acetic acids. In the oxidative part of the fermentation, the hexose is converted to 2 acetic acids and 2 CO(2) molecules with the formation of 2 NADH and 2 reduced ferredoxin (Fd(red)(2-)) molecules. In the reductive part, 2 CO(2) molecules are reduced to acetic acid, consuming the 8 reducing equivalents generated in the oxidative part. An open question is how the two parts are electronically connected, since two of the four oxidoreductases involved in acetogenesis from CO(2) are NADP specific rather than NAD specific. We report here that the 2 NADPH molecules required for CO(2) reduction to acetic acid are generated by the reduction of 2 NADP(+) molecules with 1 NADH and 1 Fd(red)(2-) catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase (NfnAB). The cytoplasmic iron-sulfur flavoprotein was heterologously produced in Escherichia coli, purified, and characterized. The purified enzyme was composed of 30-kDa (NfnA) and 50-kDa (NfnB) subunits in a 1-to-1 stoichiometry. NfnA harbors a [2Fe2S] cluster and flavin adenine dinucleotide (FAD), and NfnB harbors two [4Fe4S] clusters and FAD. M. thermoacetica contains a second electron-bifurcating enzyme. Cell extracts catalyzed the coupled reduction of NAD(+) and Fd with 2 H(2) molecules. The specific activity of this cytoplasmic enzyme was 3-fold higher in H(2)-CO(2)-grown cells than in glucose-grown cells. The function of this electron-bifurcating hydrogenase is not yet clear, since H(2)-CO(2)-grown cells additionally contain high specific activities of an NADP(+)-dependent hydrogenase that catalyzes the reduction of NADP(+) with H(2). This activity is hardly detectable in glucose-grown cells.     

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 micromol. min(-1). mg(-1). The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP(+) oxidoreductase activity and catalyzed the reduction of NADP(+) with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.     

Hydrogen gas evolution and carbon dioxide fixation with visible light by chlorophyllin coupled with polyethylene glycol

Chlorophyllin a was conjugated with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene), PEG-NH(2), to form the PEG-chlorophyllin conjugate through acid-amide bonds. The PEG-chlorophyllin conjugate was stable toward light illumination under anaerobic condition in comparison with chlorophyllin a. The conjugate catalyzed the reduction of methyl viologen in the presence of 2-mercaptoethanol and the evolution of hydrogen gas in the presence of methyl viologen (an electron carrier), 2-mercaptoethanol (an electron donor) and hydrogenase (Scheme 1). Furthermore, the PEG-chlorophyllin conjugate catalyzed the photoreduction of NADP(+) or NAD(+) in the presence of ascorbate as an electron donor and ferredoxin-NADP(+) reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by the PEG-chlorophyllin conjugate under the illumination, CO(2) fixation was accomplished by the synthesis of malate (C(4)) from pyruvate (C(3)) and CO(2) in the presence of malic enzyme (Scheme 2). These reactions mentioned above did never proceed in dark or without each enzyme.     

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.     

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.     

Electron transfer between reduced methyl viologen and oxidized glutathione: a new assay of Saccharomyces cerevisiae glutathione reductase

Pure glutathione reductase from Saccharomyces cerevisiae catalyzed under anaerobic conditions the enzymatic reduction of GSSG using electrochemically reduced methyl viologen as electron donor. The new assay was completely dependent on the amount of active enzyme present, and involved the formation of 1 mol GSH per mole of reduced methyl viologen consumed. The enzyme followed a standard Michaelis-Menten kinetics; a Km = 230 microM for reduced methyl viologen and a turnover number of 969 mumol GSSG reduced per minute per micromole enzyme were determined. The enzymatic activity seemed to depend on the redox potential, showing half-maximal activity at -0.407 V. The enzyme was quite specific: the activity using reduced benzyl viologen as electron donor was just 1.5% of that obtained with reduced methyl viologen at the same concentration and potential. Glutathione reductase was totally inactivated after a brief anaerobic exposure with reduced methyl viologen in the absence of GSSG; a partial reactivation was observed following addition of glutathione disulfide. No inhibition of the methyl viologen-dependent activity was observed in the presence of 2',5'-ADP or 2'-P-5'-ADP-ribose, two NADP(H) analogs, at concentrations which drastically inhibited the NADPH-dependent activity, thus suggesting that the reduced viologen does not interact with the pyridine nucleotide-binding site.     

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.     

Purification and characterization of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from a nitrogen-fixing bacterium

Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP(+) reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP(+) reductase in the photochemical reduction of NADP(+) by blue-green algal particles. The ferredoxin-NADP(+) reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP(+) was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD(+) transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (K(m) = 5.0 x 10(-3)M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase.     

Identification and characterization of oxalate oxidoreductase, a novel thiamine pyrophosphate-dependent 2-oxoacid oxidoreductase that enables anaerobic growth on oxalate

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 μM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.     

Influence of nitrate on oxalate- and glyoxylate-dependent growth and acetogenesis by Moorella thermoacetica

Oxalate and glyoxylate supported growth and acetate synthesis by Moorella thermoacetica in the presence of nitrate under basal (without yeast extract) culture conditions. In oxalate cultures, acetate formation occurred concomitant with growth and nitrate was reduced in the stationary phase. Growth in the presence of [(14)C]bicarbonate or [(14)C]oxalate showed that CO(2) reduction to acetate and biomass or oxalate oxidation to CO(2) was not affected by nitrate. However, cells engaged in oxalate-dependent acetogenesis in the presence of nitrate lacked a membranous b-type cytochrome, which was present in cells grown in the absence of nitrate. In glyoxylate cultures, growth was coupled to nitrate reduction and acetate was formed in the stationary phase after nitrate was totally consumed. In the absence of nitrate, glyoxylate-grown cells incorporated less CO(2) into biomass than oxalate-grown cells. CO(2) conversion to biomass by glyoxylate-grown cells decreased when cells were grown in the presence of nitrate. These results suggest that: (1) oxalate-grown cells prefer CO(2) as an electron sink and bypass the nitrate block on the acetyl-CoA pathway at the level of reductant flow and (2) glyoxylate-grown cells prefer nitrate as an electron sink and bypass the nitrate block of the acetyl-CoA pathway by assimilating carbon via an unknown process that supplements or replaces the acetyl-CoA pathway. In this regard, enzymes of known pathways for the assimilation of two-carbon compounds were not detected in glyoxylate- or oxalate-grown cells.     

Oxidoreductases Involved in Cell Carbon Synthesis of Methanobacterium thermoautotrophicum

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60°C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; α-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent V_max and K_M values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective α-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the α-ketoacid and CO₂. The data indicate that the two enzymes are similar to pyruvate synthase and α-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed.     

Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis

Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase.     

Purification and properties of a NADH-dependent 5,10-methylenetetrahydrofolate reductase from Peptostreptococcus productus

The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 mumols.min-1 mg protein-1 (37 degrees C; pH 5.5). The apparent Km for NADH was near 10 microM. The apparent molecular mass of the enzyme was determined by gel filtration to be approximately 250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used to substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP(+)-dependent reductase of eucaryotes investigated so far.     

CO oxidoreductase from Streptomyces strain G26 is a molybdenum hydroxylase.

CO oxidoreductase was purified to 95% homogeneity from crude mycelial extracts of Streptomyces G26. The purified preparation has a specific activity of 25.7 units/mg, a 13-fold improvement on crude soluble mycelial extracts. The native enzyme (Mr 282,000) is composed of non-identical subunits of Mr 110,000 and 33,000. It is a molybdenum hydroxylase containing 1.6 mol of FAD, 7.3 mol of Fe, 8.3 mol of acid-labile sulphide and 1.3 mol of Mo per mol of enzyme. Purified CO oxidoreductase catalyses the reduction of benzyl viologen, confirming the previously reported ability of this enzyme to interact with low-potential acceptors. Cytochrome c reduction cannot be accounted for entirely by non-enzymic reduction by superoxide radicals. NAD+ and NADP+ are not reduced, nor is clostridial ferredoxin.     

Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of "Desulfomonile tiedjei"

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, "Desulfomonile tiedjei." We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c(3), or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, CO, or H(2), but not by pyruvate plus coenzyme A or by dithionite. The pH and temperature optima for aryl dehalogenation were 8.2 and 35 degrees C, respectively. The rate of dehalogenation was proportional to the amount of protein in the assay mixture. The substrate specificity of aryl dehalogenation activity for various aromatic compounds in "D. tiedjei" cell extracts was identical to that of whole cells, except differences were observed in the relative rates of halobenzoate transformation. Dehalogenation was 10-fold greater in "D. tiedjei" extracts prepared from cells cultured in the presence of 3-chlorobenzoate, suggesting that the activity was inducible. Aryl reductive dehalogenation in extracts was inhibited by sulfite, sulfide, and thiosulfate, but not sulfate. Experiments with combinations of substrates suggested that cell extracts dehalogenated 3-iodobenzoate more readily than either 3,5-dichlorobenzoate or 3-chlorobenzoate. Dehalogenation activity was found to be membrane associated. This is the first report characterizing aryl dehalogenation activity in cell extracts of an obligate anaerobe.     

Demonstration of hydrogenase in extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum.

Cell-free extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum were shown to catalyze the hydrogen-dependent reduction of various artificial electron acceptors. The activity of the hydrogenase was optimal at pH 8.5 to 9 and was extremely sensitive to aeration. EDTA did not significantly reduce the liability of the enzymic activity to oxidation (aeration). At 50 degrees C, when both methyl viologen and hydrogen were at saturating concentrations with respect to hydrogenase, the specific activity of cell-free extracts approximated 4 mumol of H2 oxidized per min per mg of protein; fourfold higher specific activities were obtained when benzyl viologen was utilized as an electron acceptor. Activity stains of polyacrylamide gels demonstrated the presence of a single hydrogenase band, suggesting that the catalytic activity in cell extracts was due to a single enzyme. The activity was stable for at least 32 min at 55 degrees C but was slowly inactivated at 70 degrees C. NAD, NADP, flavin adenine dinucleotide, flavin mononucleotide, and ferredoxin were not significantly reduced, but possible reduction of the particulate b-type cytochrome of C. thermoaceticum was observed. NaCl, sodium dodecyl sulfate, iodoacetamide, and CO were shown to inhibit catalysis. A kinetic study is presented, and the possible physiologic roles for hydrogenase in C. thermoaceticum ar discussed.     

Reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA in a denitrifying, phenol-degrading Pseudomonas species

The initial reactions in anaerobic degradation of phenol to CO2 have been studied in vitro with a denitrifying Pseudomonas strain grown with phenol and nitrate in the absence of molecular oxygen. Phenol has been proposed to be carboxylated to 4-hydroxybenzoate [(1987) Arch. Microbiol. 148, 213-217]. 4-Hydroxybenzoate was activated to 4-hydroxybenzoyl-CoA by a coenzyme A ligase. Cell extracts also catalyzed the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA with reduced benzyl viologen as electron donor. This enzyme, benzoyl-CoA:(acceptor) 4-oxidoreductase (hydroxylating) (EC 1.3.99.-), has not been reported before. The data suggest that phenol and 4-hydroxybenzoate are anaerobically metabolized by this strain via benzoyl-CoA.     

Purification and properties of NADP-reductase of phototropic bacteria Thiocapsa roseopersicina]

The method of purification up to homogenous states and properties of NADP-reductase of purple bacteria Thiocapsa roseopersicina, strain BBS, are described. The molecular weight of NADP-reductase is about 47 000; it is flavoprotein consisting of two subunits. Atebrim and chloromercury bensoate inhibit the activity of NADP-reductase (34% and 33--60%, respectively). The enzyme is specific to NADPH; it catalyzes menadion-reductase reaction, diaphorase reaction of benzyl viologen reduction, oxidation of reduced benzyl viologen in the presence of NADP, reduction of ferredoxin and cytochrome c in the presence of NADPH, but it is not capable to catalyze transhydrogenase reaction.     

Partial purification of ferredoxin from Ruminococcus albus and its role in pyruvate metabolism and reduction of nicotinamide adenine dinucleotide by H2.

Extracts of Ruminococcus albus were not able to convert pyruvate to acetyl phosphate, CO2, and H2 after passage through a diethylaminoethyl (DEAE)-cellulose column. Activity was restored by a brown protein fraction eluted from the column with 0.4 M Cl-. The protein was partially purified and shown to have the spectral and biological characteristics of ferredoxin. R. albus ferredoxin, Clostridium pasteurianum ferredoxin, and methyl viologen restored activity for pyruvate decomposition by DEAE-cellulose-treated R. albus extracts. R. albus or C. pasteurianum ferredoxin restored the ability of DEAE-cellulose-treated C. pasteurianum extracts to form H2 and acetyl phosphate from pyruvate. Ferredoxin-free extracts of R. albus reduced nicotinamide adenine dinucleotide (NAD) when supplemented with R. albus or C. pasteurianum ferredoxin or with methyl viologen. These extracts reduced NADP with H2 poorly unless both ferredoxin and NAD were added, which indicates the presence of an NADH:NADP transhydrogenase. Flavin mononucleotide and flavin adenine dinucleotide were rapidly reduced by H2 by ferredoxin-free extracts in the absence of ferredoxin.     

Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).