Electrochemical characterization of a truncated hydrogenase from Pyrococcus furiosus

Abstract A highly thermostable cytoplasmic [NiFe] hydrogenase PfSHI, incorporating four subunits (αβγδ), displays a clear direct electron transfer (DET) type bioelectrocatalytic activity. In order to investigate the biological and electrochemical properties of the corresponding truncated hydrogenases, we constructed two truncated enzymes (i.e. Pfαδ and Pfα) derived from the native hydrogenase and immobilized them on multiwalled carbon nanotubes casted glassy carbon electrodes. The cyclic voltammogram along with the enzymatic activity assay revealed that the dimer Pfαδ showed an obvious oxidation current under H2 in a wide temperature range, demonstrating its DET capability, while the monomer Pfα had no activity in H2 oxidation. At high potentials, Pfαδ was proved to be inactive and observed no reverse recovery on the return scan under 40 to 60°C. Notably, Pfαδ still maintained oxygen tolerance to some extent and its activity can be recovered after applying a reduction potential. Further, enzyme turnover frequencies and kinetic parameters were analyzed based on a steady state model, which revealed that Pfαδ exhibited a higher electron transfer rate than that of pristine PfSHI due to the shortened electron transfer distance between the enzyme and the electrode.