Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.     

Formate dehydrogenase molybdenum and tungsten sites--observation by EXAFS of structural differences

Preliminary EXAFS data has been collected on the molybdenum (K-edge) in C. pasteurianum formate dehydrogenase and the tungsten (LIII-edge) in C. thermoaceticum formate dehydrogenase. In the presence of dithionite, the tungsten enzyme was devoid of W = O bonds, and exhibited average W-(O, N) and W-S bond lengths of 2.13 +/- 0.03 A and 2.39 +/- 0.03 A, respectively. In sharp contrast, the C. pasteurianum molybdenum site has three Mo = O bonds with an average bond length of 1.74 +/- 0.03 A. It is also the first molybdenum enzyme found lacking Mo-S bonds, and does not appear to be redox active in the presence of formate or dithionite. Model compounds WO2(8-hydroxyquinoline)2 = WO2(ox)2, and WO2(8 mercaptoquinoline)2 = WO2(tox)2, were also examined. Respective predicted bond lengths for WO2(ox)2 and WO2(tox)2 were W = O of 1.71, 1.73 A; W-N of 2.31, 2.29 A; W-O or W-S of 1.92 or 2.40 A, with estimated uncertainties of +/- 0.03 A.     

Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies.

Azospirillum brasilense glutamate synthase has been studied by absorption, electron paramagnetic resonance, and circular dichroism spectroscopies in order to determine the type and number of iron-sulfur centers present in the enzyme alpha beta protomer and to gain information on the role of the flavin and iron-sulfur centers in the catalytic mechanism. The FMN and FAD prosthetic groups are demonstrated to be non-equivalent with respect to their reactivities with sulfite. Sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct with a Kd of approximately 1 mM. The enzyme-sulfite complex is reduced by NADPH, and the complexed sulfite is competitively displaced by 2-oxoglutarate, which suggests the reactive flavin to be at the imine-reducing site. These data are in agreement with the two-site model of the enzyme active center proposed on the basis of kinetic studies [Vanoni, M.A., Nuzzi, L., Rescigno, M., Zanetti, G., & Curti, B. (1991) Eur. J. Biochem. 202, 181-189]. Each enzyme protomer was found, by chemical analysis, to contain 12.1 +/- 0.5 mol of non-heme iron. Electron paramagnetic resonance spectroscopic studies on the oxidized and reduced forms of glutamate synthase demonstrated the presence of three distinct iron-sulfur centers per enzyme protomer. The oxidized enzyme exhibits an axial spectrum with g values at 2.03 and 1.97, which is highly temperature-dependent and integrates to 1.1 +/- 0.2 spin/protomer. This signal is assigned to a [3Fe-4S]1+ cluster (Fe-S)I. Reduction of the enzyme with an NADPH-regenerating system results in reduction of the [3Fe-4S]1+ center to a species with a g approximately 12 signal characteristic of the S = 2 spin state of a [3Fe-4S]0 cluster. The NADPH-reduced enzyme also exhibits an [Fe-S] signal at g values of 1.98, 1.95, and 1.88, which integrates to 0.9 spin/protomer and is due to a second cluster (Fe-S)II. Reduction of the enzyme with the light/deazaflavin method results in a signal characteristic of [Fe-S] clusters with g values of 2.03, 1.92, and 1.86 and an integrated intensity of 1.9 spin/protomer. This signal arises from reduction of the (Fe-S)II center and from that of the third, lower potential iron-sulfur center (Fe-S)III. Circular dichroism spectral data on the oxidized and reduced forms of the enzyme are more consistent with the assignment of (Fe-S)II and (Fe-S)III as [4Fe-4S] clusters rather than [2Fe-2S] centers.     

Inhibition of xanthine oxidase and xanthine dehydrogenase by nitric oxide. Nitric oxide converts reduced xanthine-oxidizing enzymes into the desulfo-type inactive form

Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were inactivated by incubation with nitric oxide under anaerobic conditions in the presence of xanthine or allopurinol. The inactivation was not pronounced in the absence of an electron donor, indicating that only the reduced enzyme form was inactivated by nitric oxide. The second-order rate constant of the reaction between reduced XO and nitric oxide was determined to be 14.8 +/- 1.4 M-1 s-1 at 25 degrees C. The inactivated enzymes lacked xanthine-dichlorophenolindophenol activity, and the oxypurinol-bound form of XO was partly protected from the inactivation. The absorption spectrum of the inactivated enzyme was not markedly different from that of the normal enzyme. The flavin and iron-sulfur centers of inactivated XO were reduced by dithionite and reoxidized readily with oxygen, and inactivated XDH retained electron transfer activities from NADH to electron acceptors, consistent with the conclusion that the flavin and iron-sulfur centers of the inactivated enzyme both remained intact. Inactivated XO reduced with 6-methylpurine showed no "very rapid" spectra, indicating that the molybdopterin moiety was damaged. Furthermore, inactivated XO reduced by dithionite showed the same slow Mo(V) spectrum as that derived from the desulfo-type enzyme. On the other hand, inactivated XO reduced by dithionite exhibited the same signals for iron-sulfur centers as the normal enzyme. Inactivated XO recovered its activity in the presence of a sulfide-generating system. It is concluded that nitric oxide reacts with an essential sulfur of the reduced molybdenum center of XO and XDH to produce desulfo-type inactive enzymes.     

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.     

Formate dehydrogenase from the methane oxidizer Methylosinus trichosporium OB3b.

Formate dehydrogenase (NAD+ dependent) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isolated anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 cellulose and Sephacryl S-300 columns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the alpha-subunit was 53,800 +/- 2,800, and that of the beta-subunit was 102,600 +/- 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent alpha 2 beta 2 arrangement. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were detected. No other metals were found in stoichiometric amounts. When the enzyme was isolated aerobically, there was no cofactor requirement for NAD reduction; however, when isolated anaerobically, activity was 80 to 90% dependent on the addition of flavin mononucleotide (FMN) to the reaction mixture. Furthermore, the addition of formate to an active, anoxic solution of formate dehydrogenase rapidly inactivated it in the absence of an electron acceptor; this activity could be reconstituted approximately 85% by 50 nM FMN. Flavin adenine dinucleotide could not replace FMN in reconstituting enzyme activity. The Kms of formate dehydrogenase for formate, NAD, and FMN were 146, 200, and 0.02 microM, respectively. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inactivated under anoxic conditions by formate and reactivated by FMN. The evolutionary significance of this similarity is discussed.     

The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.

The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.     

Action mechanism of Escherichia coli DNA photolyase. III. Photolysis of the enzyme-substrate complex and the absolute action spectrum

The absolute action spectrum of Escherichia coli DNA photolyase was determined in vitro. In vivo the photoreactivation cross-section (epsilon phi) is 2.4 X 10(4) M-1 cm-1 suggesting that the quantum yield (phi) is about 1.0 if one assumes that the enzyme has the same spectral properties (e.g. epsilon 384 = 1.8 X 10(4) M-1 cm-1) in vivo as those of the enzyme purified to homogeneity. The relative action spectrum of the pure enzyme (blue enzyme that contains FAD neutral semiquinone radical) agrees with the relative action spectrum for photoreactivation of E. coli, having lambda max = 384 nm. However, the absolute action spectrum of the blue enzyme yields a photoreactivation cross-section (epsilon phi = 1.2 X 10(3) at 384 nm) that is 20-fold lower than the in vivo values indicative of an apparent lower quantum yield (phi approximately equal to 0.07) in vitro. Reducing the enzyme with dithionite results in reduction of the flavin semiquinone and a concomitant 12-15-fold increase in the quantum yield. These results suggest that the flavin cofactor of the enzyme is fully reduced in vivo and that, upon absorption of a single photon in the 300-500 nm range, the photolyase chromophore (which consists of reduced FAD plus the second chromophore) donates an electron to the pyrimidine dimer causing its reversal to two pyrimidines. The reduced chromophore is regenerated at the end of the photochemical step thus enabling the enzyme to act catalytically.+     

Characterization of a flavoprotein iodotyrosine deiodinase from bovine thyroid. Flavin nucleotide binding and oxidation-reduction properties.

A stable apoprotein has been prepared from a soluble purified bovine thyroid iodotyrosine deiodinase, previously shown to be an FMN-containing flavoprotein requiring dithionite for enzymatic activities. The apoprotein binds FMN (Ka = 1.47 x 10(8) M-1) with an almost complete restoration of enzymatic activity. It can also bind FAD (Ka = 0.58 x 10(8) M-1) with partial restoration of activity, but does not bind riboflavin. Photoreduction of the holoenzyme in presence of excess of its free cofactor, FMN, supported enzyme activity at a level of 50% of that obtained with dithionite; substituting FAD or riboflavin for FMN produced, respectively, 20 and 11% of the dithionite-supported activity. The oxidation-reduction potential (E1) of the couple semiquinone/fully reduced enzyme is -0.412 V at pH 7 and 25 degrees C. The value (E2) for the oxidized/semiquinone couple is -0.190 V at pH 7 and 25 degrees C. Potentiometric titrations with sodium hydrosulfite suggests that the enzyme is reduced in two successive 1-electron oxidation-reduction steps. Effects of pH on E1 suggest ionization of the protonated flavin with an ionization constant of 5.7 x 10(-7). The highly negative oxidation-reduction potential for the fully reduced enzyme species and the apparent requirement for full reduction for enzymatic activity suggests that in NADPH-mediated microsomal deiodination an NADPH-linked electron carrier of suitably negative midpoint potential is a probable intermediate.     

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

Reduced diphosphopyridine nucleotide peroxidase. Intermediates formed on reduction of the enzyme with dithionite or reduced diphosphopyridine nucleotide.

DPNH peroxidase is a flavin adenine dinucleotide-containing flavoprotein. Anaerobic titration of enzyme with dithionite has shown that the active site of the enzyme contains 2 mol of flavin and in addition 1 mol of a non-flavin electron acceptor that is tentatively identified as a disulfide group. Thus complete reduction of the enzyme requires 3 mol of dithionite per mole of active site. The first mole of dithionite reduces the non-flavin acceptor; complex formation between the reduced acceptor and one of the bound flavin molecules causes the formation of a long wavelength absorption band between 500 and 670 nm. The second mole of dithionite reduces the flavin that interacts with the reduced non-flavin group, and the long wavelength band disappears. The third mole of dithionite reduces the second mole of flavin. All groups are reoxidized in the presence of air. DPNH reacts with only two of the enzyme-bound electron acceptors. The first mole of DPNH reduces the non-flavin group to form an intermediate (I) that is almost identical with that formed by dithionite. The second mole of DPNH complexes with the second flavin of Intermediate I to form Intermediate II. This reaction causes a further absorbance increase in the long wavelength region; the tail of the absorption band now extends to 960 nm. The titration data (potassium phosphate, 0.05 M, pH 7.0) can be fitted with dissociation constants of 1 times 10-7 M for the formation of I, and 3 times 10-6 M for the conversion of I to II. In air, species II is oxidized to I; I is stable in air, but is oxidized stoichiometrically to oxidized enzyme by H2O2. Present evidence suggests that bound DPN-plus is responsible for the air stability of species I. Intermediate I, but not oxidized enzyme, reacts slowly with phenylmercuric acetate. This reaction causes loss of the air-stable intermediate and parallel loss in enzyme activity. The inactive enzyme cannot be reduced by DPNH to Species I; DPNH can, however, still react with the second flavin to form the autoxidizable complex. With other methods of enzyme inactivation there is also a direct correlation between residual enzyme activity and the ability of enzyme to form the air-stable intermediate. It is concluded that the air-stable intermediate is an important catalytic species.     

Purification and properties of the flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii.

A pyridine nucleotide independent D-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975 J. Bacteriol. 124, 1454--1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for D-lactate, and no inhibition is observed with L-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a gamma/2 of 0.224 and 0.442, respectively. Except for this atypically high oxygen reactivity, D-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by D-lactate. Two electrons per flavin were consumed in a dithionite titration, implyine with varying ratios of D-lactate and pyruvate, an Em7 of -0.219 +/- 0.007 V at 20 degrees C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivation results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation.     

Purification and characterization of glutamate synthase from Azospirillum brasilense.

Growth conditions for Azospirillum brasilense Sp6 were devised for maximal expression of glutamate synthase. The enzyme levels were largely affected by the type and concentration of the nitrogen source. A 10-fold increase in the synthesis of the enzyme was observed at a limiting concentration of ammonia. The enzyme was purified to homogeneity by a procedure which was fairly rapid and allowed a good recovery of enzyme (30%). Azospirillum glutamate synthase is a complex iron-sulfur flavoprotein with a stoichiometry of 1 flavin adenine dinucleotide:1 flavin mononucleotide:8 Fe:8 S per protomer with a molecular weight of 185,000. The protomer is composed of two dissimilar subunits with molecular weights of 135,000 and 50,000. Kinetic parameters were determined. Km values for NADPH, 2-oxoglutarate, and L-glutamine were 6.25, 29, and 450 microM, respectively. The optimum pH was about 7.5. Complete reduction of the enzyme under anaerobic conditions was obtained either by NADPH (in the presence of a regenerating system) or dithionite or by photochemical reduction (in the presence of EDTA and 5-deazariboflavin). No stable long-wavelength intermediates were observed.     

The interaction of bisulfite with milk xanthine oxidase

Bisulfite ion competitively inhibits xanthine oxidase activity. The ability of HSO3- to bind at the molybdenum center is controlled by pH due to a pKa of 6.91 for SO3(2-)/HSO3-. The Kd for the enzyme-bisulfite complex is 4.5 x 10(-5) M at pH 7.0 and 25 degrees C. The relative magnitude of extinction changes in the optical absorption spectra, the number of inhibitor ions reversibly bound, and the number of electrons required for complete bleaching of the visible spectrum of the milk xanthine oxidase-HSO3- complex were all dependent on the percentage of fully functional xanthine oxidase. Binding of HSO3- causes perturbations of the visible spectrum: the maximum extinction changes at 320 and 422 nm were calculated to be -4300 and -2150 M-1 cm-1, respectively. The stoichiometry of reversible binding was determined to be one molecule of HSO3-/active molybdenum center. Combined optical and EPR analyses of anaerobic dithionite titrations revealed that the relative redox potentials of the Mo6+/5+ and Mo5+/4+ couples decreased by approximately 35 and 45 mV on binding bisulfite, respectively. The finding that bisulfite has a profound effect on the redox properties of xanthine oxidase necessitates a re-evaluation of dithionite titrations previously carried out with this enzyme at neutral and low pH values since bisulfite produced as an oxidation product of dithionite binds to the enzyme during the course of titration.     

Cytochrome b from Escherichia coli nitrate reductase. Its properties and association with the enzyme complex

Membrane-bound nitrate reductase purified from Escherichia coli was resolved into two separate forms. The majority of the enzyme complex had a subunit composition of 2A:2B:4C, exhibited cytochrome b spectra, and was found to be stable after purification. A second form of nitrate reductase activity was a modified complex with a subunit composition of 2A:2B and lacked cytochrome. The subunit B from this complex was altered in its mobility on sodium dodecyl sulfate-polyacrylamide gels. The cytochrome-containing enzyme had 28 +/- 2 atoms of iron and 1.35 atoms of molybdenum whereas iron and molybdenum in cytochromeless enzyme were 24 +/- 2 atoms and 1.18 atoms/molecule, respectively. Besides cytochrome-containing nitrate reductase, two other cytochrome b-containing fractions were also resolved. These were cytochrome b associated with formate dehydrogenase and a novel cytochrome b with reduced absorption maxima at 430, 529.5, and 560 nm. Nitrate reductase cytochrome b (subunit C) was isolated from subunits A and B as a partially denatured form and its renaturation was accomplished by dialyzing against hemin. The renatured cytochrome yielded absorption spectra similar to the holoenzyme. The pure cytochrome aggregated upon heating, even in the presence of sodium dodecyl sulfate. It had a high isoelectric point (pH greater than 9.5) and had 45% hydrophobic amino acids.     

Tuning a nitrate reductase for function. The first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S](1+) cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E(m)) of -190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g(av) = 2.0047 in air-oxidized enzyme that accounted for only 10-20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g(av) = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of -100 to -350 mV (E(m) Mo(6+/5+) = -150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below -200 mV, where the cofactors are predominantly [4Fe-4S](1+) and Mo(5+). The data suggests that during the catalytic cycle nitrate will bind to the Mo(5+) state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.     

On the redox properties of three bisulfite reductases from the sulfate-reducing bacteria.

The redox properties of purified bisulfite reductases from Desulfovibrio gigas, D. desulfuricans (Norway) and Desulfotomaculum ruminis, containing non-heme iron and siroheme have been studied by EPR spectroscopy. Each enzyme shows ferric siroheme EPR signals which are not completely reduced by dithionite after 20 min, but are readily reduced within 1 min by dithionite plus methyl viologen. With the latter reducing system, each reductase also reveals a variable Beinert “g=1.94” type iron-sulfur signal. Reaction of each reductase with reduced methyl viologen results in reduction of only the siroheme. These results suggest different redox potentials for the iron-sulfur and siroheme moieties, and indicate that their functional properties are similar for each reductase.     

Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS.

The two components of the inducible 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS were purified to homogeneity. Yellow component B is a monomer (Mr, 37,500) with NADH-acceptor reductase activity. Ferricyanide, 2,6-dichlorophenol indophenol, and cytochrome c acted as electron acceptors. Component B was identified as an iron-sulfur flavoprotein containing 0.8 mol of flavin adenine dinucleotide, 1.7 mol of iron, and 1.7 mol of acid-labile sulfide per mol of enzyme. The isoelectric point was estimated to be pH 4.2. Component B was reduced by the addition of NADH. Red-brown component A (Mr, 200,000 to 220,000) is an iron-sulfur protein containing 5.8 mol of iron and 6.0 mol of acid-labile sulfide. The isoelectric point was within the range of pH 4.5 to 5.3. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. Component A consisted of nonidentical subunits alpha (Mr, 52,000) and beta (Mr, 20,000). It contained approximately equimolar amounts of alpha and beta, and cross-linking studies suggested an alpha 3 beta 3 subunit structure of component A. The NADH- and Fe(2+)-dependent enzyme system was named 2-halobenzoate 1,2-dioxygenase, because it catalyzes the conversion of 2-fluoro-, 2-bromo-, 2-chloro-, and 2-iodobenzoate to catechol. 2-Halobenzoate 1,2-dioxygenase exhibited a very broad substrate specificity, but benzoate analogs with electron-withdrawing substituents at the ortho position were transformed preferentially.