Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H-driven NAD-reduction in the presence of O.

Biocatalysts that mediate the H-dependent reduction of NAD to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD-reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD-reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1 (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H-mediated NAD reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD-reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H-driven cofactor recycling under oxic conditions at elevated temperatures.