A kinetic and thermodynamic understanding of O ₂ tolerance in [NiFe]-hydrogenases

<jats:p> In biology, rapid oxidation and evolution of H <jats:sub>2</jats:sub> is catalyzed by metalloenzymes known as hydrogenases. These enzymes have unusual active sites, consisting of iron complexed by carbonyl, cyanide, and thiolate ligands, often together with nickel, and are typically inhibited or irreversibly damaged by O <jats:sub>2</jats:sub> . The Knallgas bacterium <jats:italic>Ralstonia eutropha</jats:italic> H16 ( <jats:italic>Re</jats:italic> ) uses H <jats:sub>2</jats:sub> as an energy source with O <jats:sub>2</jats:sub> as a terminal electron acceptor, and its membrane-bound uptake [NiFe]-hydrogenase (MBH) is an important example of an “O <jats:sub>2</jats:sub> -tolerant” hydrogenase. The mechanism of O <jats:sub>2</jats:sub> tolerance of <jats:italic>Re</jats:italic> MBH has been probed by measuring H <jats:sub>2</jats:sub> oxidation activity in the presence of O <jats:sub>2</jats:sub> over a range of potential, pH and temperature, and comparing with the same dependencies for individual processes involved in the attack by O <jats:sub>2</jats:sub> and subsequent reactivation of the active site. Most significantly, O <jats:sub>2</jats:sub> tolerance increases with increasing temperature and decreasing potentials. These trends correlate with the trends observed for reactivation kinetics but not for H <jats:sub>2</jats:sub> affinity or the kinetics of O <jats:sub>2</jats:sub> attack. Clearly, the rate of recovery is a crucial factor. We present a kinetic and thermodynamic model to account for O <jats:sub>2</jats:sub> tolerance in <jats:italic>Re</jats:italic> MBH that may be more widely applied to other [NiFe]-hydrogenases. </jats:p>