Catalytic Production of Hydrogen Peroxide and Water by Oxygen-Tolerant [NiFe]-Hydrogenase during H2 Cycling in the Presence of O2

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Hydrogenases control the H2-related metabolism in many microbes. Most of these enzymes are prone to immediate inactivation by O2. However, a few members of the subclass of [NiFe]-hydrogenases are able to convert H2 into protons and electrons even in the presence of O2, making them attractive for biotechnological application. Recent studies on O2-tolerant membrane-bound hydrogenases indicate that the mechanism of O2 tolerance relies on their capability to completely reduce O2 with four electrons to harmless water. In order to verify this hypothesis, we probed the O2 reduction capacity of the soluble, NAD(+)-reducing [NiFe]-hydrogenase (SH) from Ralstonia eutropha H16. A newly established, homologous overexpression allowed the purification of up to 90 mg of homogeneous and highly active enzyme from 10 g of cell material. We showed that the SH produces trace amounts of superoxide in the course of H2-driven NAD(+) reduction in the presence of O2. However, the major products of the SH-mediated oxidase activity was in fact hydrogen peroxide and water as shown by the mass spectrometric detection of H2(18)O formed from H2 and isotopically labeled (18)O2. Water release was also observed when the enzyme was incubated with NADH and (18)O2, demonstrating the importance of reverse electron flow to the [NiFe] active site for O2 reduction. A comparison of the turnover rates for H2 and O2 revealed that in the presence of twice the ambient level of O2, up to 3% of the electrons generated through H2 oxidation serve as "health insurance" and are reused for O2 reduction.