Publications

• Functional analysis by site-directed mutagenesis of the NAD(+)-reducing hydrogenase from Ralstonia eutropha.

  Burgdorf T., De Lacey A.L., Friedrich B.

  The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD(+) under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site ... >> More

  The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD(+) under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H(2)-oxidizing activity. In one of these isolates (HoxH I64A), H(2) binding was impaired. Class II mutants revealed a high D(2)/H(+) exchange rate relative to a low H(2)-oxidizing activity. A representative (HoxH H16L) displayed D(2)/H(+) exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O(2)-sensitive growth on hydrogen due to an O(2)-sensitive SH protein. << Less

  J Bacteriol 184:6280-6288(2002) [PubMed] [EuropePMC]

• Effect of redox potential on the activation of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1.

  Petrov R.R., Utkin I.B., Popov V.O.

  A formal kinetic treatment of the autocatalytic activation cycle of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1 is presented. The value for the enzyme first-order activation rate constant is estimated to be (2.0 +/-0.6) s-1 (pH 7.8, 25 degrees C). The effect of the redox potential ... >> More

  A formal kinetic treatment of the autocatalytic activation cycle of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1 is presented. The value for the enzyme first-order activation rate constant is estimated to be (2.0 +/-0.6) s-1 (pH 7.8, 25 degrees C). The effect of the redox potential on the activation properties of the NAD-dependent hydrogenase is studied. Hydrogenase activation is controlled by a midpoint redox potential of approximately -100 mV (pH 7.8). Once activated the enzyme is not immediately transformed back into an inactive state on rapid reoxidation and is able to preserve its catalytic properties for at least 3-4 h of intense oxigenation. Several lines of evidence show that the reductive activation of the NAD-dependent hydrogenase is accompanied by a structural reorganization of the protein. A possible origin of the -100 mV transition is discussed. A model for the activation process of the NAD-dependent hydrogenase is suggested. << Less

  Arch Biochem Biophys 268:287-297(1989) [PubMed] [EuropePMC]

• Characterization of hydrogenase II from the hyperthermophilic archaeon Pyrococcus furiosus and assessment of its role in sulfur reduction.

  Ma K., Weiss R., Adams M.W.

  The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (alphabetagammadelta), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H(2) production and the reduction of elemental sulfur to H(2)S. Herein is described the purification of a second en ... >> More

  The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (alphabetagammadelta), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H(2) production and the reduction of elemental sulfur to H(2)S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an M(r) of 320,000 +/-20,000 and contains four different subunits with M(r)s of 52,000 (alpha), 39,000 (beta), 30,000 (gamma), and 24,000 (delta). The heterotetramer contained Ni (0.9 +/- 0.1 atom/mol), Fe (21 +/-1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 +/-0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H(2) production with K(m) values near 70 microM and k(cat)/K(m) values near 350 min(-1) mM(-1). In contrast to hydrogenase I, hydrogenase II catalyzed the H(2)-dependent reduction of NAD (K(m), 128 microM; k(cat)/K(m), 770 min(-1) mM(-1)). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H(2) and NADPH served as electron donors for the reduction of elemental sulfur (S(0)) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H(2) using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the beta subunit and three in the delta subunit) and one [2Fe-2S] cluster (in the gamma subunit), as well as two putative nucleotide-binding sites in the gamma subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S(0) concentrations since it has a higher affinity than hydrogenase I for both S(0) and polysulfide. << Less

  J. Bacteriol. 182:1864-1871(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Hydrogenases I and II from Pyrococcus furiosus.

  Ma K., Adams M.W.

  Methods Enzymol. 331:208-216(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.