Activation and deactivation of the membrane-bound hydrogenase from Desulfovibrio desulfuricans, Norway strain

The hydrogenase from D. desulfuricans, when isolated in air, had a low activity in the hydrogen-methyl viologen reductase assay, and no activity in the hydrogen-methylene blue reductase assay. The activity increased markedly during incubation under hydrogen. This process is interpreted in terms of conversion of the enzyme from a relatively inactive Unready state to the Active state. Oxidation by dichloro-indophenol caused conversion to a state in which the hydrogen-uptake activity to methyl viologen was preserved, but hydrogen-methylene blue activity was not. This form is termed the Ready state. This behaviour resembles that of the hydrogenase of Desulfovibrio gigas and thus may be a widespread property of this class of hydrogenases. The electron-spin-resonance spectra of the D. desulfuricans enzyme showed the presence of [3Fe-xS] and [4Fe-4S] clusters. Spectra were also observed in the various states of activation of the enzyme. In these respects, the hydrogenase of D. desulfuricans resembles that from D. gigas, although the latter may have an additional iron-sulphur cluster.     

Purification and properties of the soluble hydrogenase from Desulfovibrio desulfuricans (strain Norway 4)

A soluble hydrogenase has been isolated from Desulfovibrio desulfuricans (strain Norway 4) grown on Postgate's medium. The enzyme differs significantly from a membrane-bound hydrogenase previously purified from the same organism grown on Starkey's medium. The enzyme consisted of two subunits of 56 kDa and 29 kDa compared with masses of 60 kDa and 27 kDa for the membrane-bound enzyme. Analysis of preparations of the soluble enzyme by various methods gave values of 5-10 iron atoms, 6 labile sulphur atoms and 0.45-0.8 nickel atom per molecule. The enzyme was unusual in that it contained selenium, in quantities equivalent to nickel. The highly purified active enzyme produced no electron-spin-resonance (ESR) signals in the oxidized state. ESR signals due to a [3Fe-xS] cluster and nickel were observed only in some of the less active fractions of the enzyme, demonstrating that neither of these ESR-detectable components is a prerequisite for hydrogenase activity. Treatment of D. desulfuricans (Norway) cells with EDTA released a minor fraction with hydrogenase activity, which might indicate the presence of a periplasmic enzyme.     

Purification and properties of L-alanine dehydrogenase from Desulfovibrio desulfuricans

The l-alanine dehydrogenase from cell-free extracts of Desulfovibrio desulfuricans was purified approximately 56-fold. The Michaelis constants for the substrates of the amination reaction and the pH optima for the reactions catalyzed by this enzyme closely agree with those reported for other l-alanine dehydrogenases. Pyruvate was found to inhibit the amination reaction. The enzyme was absolutely specific for l-alanine and nicotinamide adenine dinucleotide. Its sensitivity to para-chloromecuribenzoate suggests that sulfhydryl groups may be necessary for enzymatic activity. These extracts also contained a nicotinamide adenine dinucleotide phosphate-specific glutamic dehydrogenase which was separated from the l-alanine dehydrogenase during purification.     

Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis.

The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.     

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.     

Purification and properties of the membrane-bound hydrogenase from N2-fixing Alcaligenes latus

The nitrogen-fixing, aerobic hydrogen-oxidizing bacterium Alcaligenes latus forms hydrogenase when growing lithoautotrophically with hydrogen as electron donor and carbon dioxide as sole carbon source or when growing heterotrophically with N2 as sole nitrogen source. The hydrogenase is membrane-bound and relatively oxygen-sensitive. The enzymes formed under both conditions are identical on the basis of the following criteria: molecular mass, mobility in polyacrylamide gel electrophoresis, Km value for hydrogen (methylene blue reduction), stability properties, localization, and cross-reactivity to antibodies raised against the 'autotrophic' hydrogenase. The hydrogenase was solubilized by Triton X-100 and deoxycholate treatment and purified by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose C1-4B, DEAE-Sephacel and Matrix Gel Red A under hydrogen to homogeneity to a specific activity of 113 mumol H2 oxidized/min per mg protein (methylene blue reduction). SDS gel electrophoresis revealed two nonidentical subunits of molecular weights of 67 000 and 34 000, corresponding to a total molecular weight of 101 000. The pure enzyme was able to reduce FAD, FMN, riboflavin, flavodoxin isolated from Megasphaera elsdenii, menadione and horse heart cytochrome c as well as various artificial electron acceptors. The reversibility of the hydrogenase function was demonstrated by H2 evolution from reduced methyl viologen.     

A cobalt porphyrin containing protein reducible by hydrogenase isolated from Desulfovibrio desulfuricans (Norway)

A cobalt-porphyrin containing protein has been isolated from the sulfate-reducer $\text{Desulfovibrio desulfuricans}$ (Norway). This violet-colored protein has a molecular weight of approx. 13,000 daltons and contains 1 cobalt atom/molecule. The apo-protein was estimated to contain 104 amino-acid residues giving a molecular weight of 11,000 daltons. The UV-visible absorption spectrum of the protein exhibiting maxima at 588,418 and 280 nm with a shoulder at 550 nm is characteristic of metalloporphyrin proteins. The molar extinction coefficients of the cobalt-protein at 588, 418 and 280 nm are 31,330 , 64,670 and 17,200 respectively and its absorbance ratio $\text{A}{}_{280}\text{A}{}_{588}$ is 0.54. The protein is reduced by dithionite giving a blue-colored reduced form. Important spectral modifications of the chromophore occurred during the reduction including a shift of the Soret peak from 418 to 381 nm and a shift of the α band in the opposite direction from 588 to 593.5 nm. The Co-protein was slowly reduced by the hydrogenase from $\text{D.desulfuricans}$ under hydrogen in the presence of cytochrome C₃. The reported data suggest that the redox states of the cobalt center of this new electron carrier correspond to the Co(III) and Co(II) states.     

Purification and some properties of cytochrome C553(550) isolated from Desulfovibrio desulfuricans Norway.

A new c-type cytochrome containing a single heme group, cytochrome c_553(550) has been purified from $\text{Desulfovibrio desulfuricans}$ (Norway strain) and some of its properties have been investigated. It has an isoelectric point of 6.6 and a higher redox potential than cytochrome c₃ isolated from the same bacteria. Its molecular weight was estimated to be 9,200 by gel filtration. The main absorption peaks are at 553, 522.5 and 417 nm in the reduced form and at 690, 529, 411, 357 and 280 nm in the oxidized form. The asymmetric α band of the reduced state is similar to the one reported for socalled “split α” cytochromes c. The cytochrome contains 86 amino acid residues with 5 methionine, two cysteine and two histidine residues. The N terminal sequence of $\text{D. desulfuricans}$ Norway cytochrome c_553(550) presents no evident homology with that of $\text{Desulfovibrio vulgaris}$ Hildenborough cytochrome c₅₅₃.     

Purification of the membrane-bound hydrogenase of Escherichia coli.

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.     

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.     

Mechanism of dissimilatory sulfite reduction by Desulfovibrio desulfuricans: purification of a membrane-bound sulfite reductase and coupling iwth cytochrome c 3 and hydrogenase

The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO₃ ^-H₂S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c ₃ from D. desulfuricans. The specific activity of mSiR was 103 nmol H₂ min^-1 mg^-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO₃ ^-H₂S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations SiR sulfite reductase - mSiR sulfite reductase purified from membranes - sSiR sulfite reductase purified from the soluble fraction Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c ₃ hydrogenase, EC 1.12.2.1     

Evidence for nickel and a three-iron center in the hydrogenase of Desulfovibrio desulfuricans

Hydrogenase from Desulfovibrio desulfuricans (ATCC No. 27774) grown in unenriched and in enriched 61Ni and 57Fe media has been purified to apparent homogeneity. Two fractions of enzymes with hydrogenase activity were separated and were termed hydrogenase I and hydrogenase II. they were shown to have similar molecular weights (77,600 for hydrogenase I and 75,500 for hydrogenase II), to be composed of two polypeptide chains, and to contain Ni and non-heme iron. Because of its higher specific activity (152 versus 97) hydrogenase II was selected for EPR and M?ssbauer studies. As isolated, hydrogenase II exhibits an "isotropic" EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.3, 2.2, and 2.0. Isotopic substitution of 61Ni proves that the rhombic signal is due to Ni. Combining the M?ssbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addition, the M?ssbauer data also revealed two [4Fe-4S]2+ clusters iun each molecule of hydrogenase II. The EPR and M?ssbauer data of hydrogenase I were found to be identical to those of hydrogenase II, indicating that both enzymes have common metallic centers.     

Electrochemical study of the electron exchange between cytochrome c3 and hydrogenase from Desulfovibrio desulfuricans Norway

The kinetics of the reduction of the Desulfovibrio desulfuricans Norway cytochrome c3 by its physiological partner hydrogenase, in the presence of hydrogen, was investigated by an electrochemical method; from cyclic voltammetry experiments a value of 3 X 10(7) M-1 s-1 was obtained for the second-order rate constant. Results are discussed in terms of specific interactions between physiological partner proteins.     

Purification and characterization of low potential c cytochromes from Desulfovibrio desulfuricans membranes

Desulfovibrio desulfuricans grown in a lactate-sulfate medium produces, in addition to soluble cytochromes, c-type cytochromes which appear to be integral membrane proteins. Two cytochromes can be separated, an abundant 15 kDa cytochrome and a 22 kDa cytochrome. Both have optical spectra characteristics of c-type cytochromes. The 15 kDa cytochrome shows two n = 1 components in potentiometric redox titrations with midpoint potentials at −130 and −270 mV in the membrane; both were slightly lower in detergent-solubilized preparations. We suggest a designation of cytochrome cc_m for this species. Its properties suggest a function as a transmembrane electron carrier between hydrogen and sulfate.