Further characterization of the [Fe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757.
The properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans ATCC 7757, previously reported to be a single-subunit protein [Glick, B. R., Martin, W. G., and Martin, S. M. (1980) Can. J. Microbiol. 26, 1214-1223] were reinvestigated. The pure enzyme exhibited a molecular mass of 53.5 kDa as measured by analytical ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N-terminal residues, respectively. The N-terminal amino acid sequences of its large and small subunits, determined up to 25 residues, were identical to those of the Desulfovibrio vulgaris Hildenborough [Fe]-hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acid-labile sulfur/molecule and had E400, 52.5 mM-1.cm-1. The purified hydrogenase showed a specific activity of 62 kU/mg of protein in the H2-uptake assay, and the H2-uptake activity was higher than H2-evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its maximum activity both in the H2-uptake and 2H2/1H2 exchange reaction. The ratio of the activity of activated to as-isolated hydrogenase was approximately 3. EPR studies allowed the identification of two ferredoxin-type [4Fe-4S]1+ clusters in hydrogenase samples reduced by hydrogen. In addition, an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H2-activating site in other [Fe]-hydrogenases was detected in partially reduced samples. Molecular properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fe]-hydrogenase, similar in most respects to the well characterized [Fe]-hydrogenase from D. vulgaris Hildenborough.
Hatchikian
E C
EC
Laboratoire de Chimie Bacterienne, Centre National de la Recherche Scientifique, Marseille, France.
Forget
N
N
Fernandez
V M
VM
Williams
R
R
Cammack
R
R
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000494:pharmacology; D002248:Carbon Monoxide / Q000494:pharmacology; D002384:Catalysis; D003300:Copper; D003901:Desulfovibrio / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel / Q000032:analysis; D012643:Selenium / Q000032:analysis; D013053:Spectrophotometry; D013455:Sulfur / Q000032:analysis
D013455:Sulfur; D003300:Copper; D009532:Nickel; D002248:Carbon Monoxide; D007501:Iron; D006864:Hydrogenase; D012643:Selenium; D001205:Ascorbic Acid