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Reinvestigation of the Steady-State Kinetics and Physiological Function of the Soluble NiFe-Hydrogenase I of Pyrococcus furiosus

Daan J. van Haaster, Pedro J. Silva, Peter-Leon Hagedoorn, Jaap A. Jongejan, and Wilfred R. Hagen^*

Department of Biotechnology, Delft University of Technology, The Netherlands

Received 28 September 2007/ Accepted 10 December 2007

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°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 K_m for H₂ (20 µM) 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 µM dissolved CO). Since the K_m for NADP⁺ is 37 µM, 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.

Published ahead of print on 21 December 2007.

Present address: Department of Agriculture and Food Sciences, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands.

Present address: Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Rua Carlos da Maia 296, 4200-150 Porto, Portugal.