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J. Bacteriol. doi:10.1128/JB.01562-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A re-investigation of the steady-state kinetics and the physiological function of the soluble NiFe-hydrogenase-I of Pyrococcus furiosus

Department of Biotechnology, Delft University of Technology, The Netherlands

^* To whom correspondence should be addressed. Email: w.r.hagen{at}tudelft.nl.

	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 then known NiFe-hydrogenases, hydrogen production activity at 80°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 would seem to obliterate 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 re-investigated. In contrast to previous reports a low K_M 20 µM for H₂ was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was an order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition at 20 µM dissolved CO). With K_M 37 µM for NADP⁺ we conclude that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH thereby re-using the hydrogen produced by the membrane-bound hydrogenase in proton respiration.