title: Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus.
abstract: Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H2 evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80 degrees C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H2 production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low Km for H2 (approximately 20 microM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 microM dissolved CO). Since the Km for NADP+ is approximately 37 microM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Daan J
authors.initials: DJ
authors.lastname: van Haaster
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Silva
authors.forename: Peter-Leon
authors.initials: PL
authors.lastname: Hagedoorn
authors.forename: Jaap A
authors.initials: JA
authors.lastname: Jongejan
authors.forename: Wilfred R
authors.initials: WR
authors.lastname: Hagen
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism*; D002248:Carbon Monoxide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000378:metabolism*; D007700:Kinetics; D020027:Pyrococcus furiosus / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature
chemical_list: D019843:Archaeal Proteins; D002248:Carbon Monoxide; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase