Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H 2 -driven NAD + -reduction in the presence of O 2 .

Biocatalysts that mediate the H ₂ -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 ₂ , 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 ^T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H ₂ -oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H ₂ -mediated NAD ⁺ reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O ₂ 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 ₂ -driven cofactor recycling under oxic conditions at elevated temperatures.
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