Catalytic Production of Hydrogen Peroxide and Water by Oxygen-Tolerant [NiFe]-Hydrogenase during H2 Cycling in the Presence of O2
2013
Lauterbach, Lars | Lenz, Oliver
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 H₂¹⁸O formed from H₂ and isotopically labeled ¹⁸O₂. Water release was also observed when the enzyme was incubated with NADH and ¹⁸O₂, 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.
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