Electrochemical Characterization Of A Truncated Hydrogenase From Pyrococcus Furiosus

A highly thermostable cytoplasmic [NiFe] hydrogenase PfSHI, incorporating four subunits (alpha beta gamma delta), 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. Pf(alpha delta), and Pf(alpha)) 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 Pf(alpha delta) showed an obvious oxidation current under H-2 in a wide temperature range, demonstrating its DET capability, while the monomer Pf(alpha), had no activity in H-2 oxidation. At high potentials, Pf(alpha delta) was proved to be inactive and observed no reverse recovery on the return scan under 40 to 60 degrees C. Notably, Pf(alpha delta) 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 Pf(alpha delta) exhibited a higher electron transfer rate than that of pristine PfSHI due to the shortened electron transfer distance between the enzyme and the electrode. (C) 2021 Elsevier Ltd. All rights reserved.