Hydrogenases from the Hyperthermophilic Archaeon Pyrococcus furiosus

Hydrogenase is an electron-transfer protein and catalyses the simplest chemical redox reaction, the reversible two-electron oxidation of molecular hydrogen in aerobic and anaerobic microorganisms. A kinetic study of the hydrogen oxidation reaction by Fe-hydrogenase from Desulfovibrio vulgaris (Hildenborough) reveals a biphasic activation mechanism from as isolated, resting enzyme, via an intermediate state of relatively low activity, to maximally active enzyme. Hydrogen itself is the causative agent for the two subsequent enzyme activation processes. Kinetic-model analysis suggests that the steady-state assumption of Michaelis-Menten kinetics does not apply to this hydrogenase. The heterotetrameric, soluble NiFe-hydrogenase-I of Pyrococcus furiosus contains a complex chain of iron-sulfur prosthetic groups for electron transfer between the H2- activating NiFe site and the coenzyme-activating flavin. EPR analysis has shown that this hydrogenase is another member of the group of multiheteromeric iron-sulfur flavin enzymes with Fe/S clusters of elusive redox properties. The steady-state kinetics of the soluble NiFe-hydrogenase-I of P. furiosus has been re-investigated. These results suggest that this enzyme is functionally similar to its NAD-reducing soluble multimeric NiFe-hydrogenase congener in Ralstonia eutropha with a KM = 37 µM for H2. The soluble P. furiosus hydrogenase has KM »37 µ for NADP+. 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.