A kinetic and thermodynamic understanding of O_2 tolerance in [NiFe]-hydrogenases

摘要

In biology, rapid oxidation and evolution of H_2 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_2. The Knallgas bacterium Ralstonia eutropha H16 (Re) uses H_2 as an energy source with O_2 as a terminal electron acceptor, and its membrane-bound uptake [NiFe]-hydrogenase (MBH) is an important example of an "02-tolerant" hydrogenase. The mechanism of O_2 tolerance of Re MBH has been probed by measuring H_2 oxidation activity in the presence of O_2 over a range of potential, pH and temperature, and comparing with the same dependencies for individual processes involved in the attack by O_2 and subsequent reactivation of the active site. Most significantly, O_2 tolerance increases with increasing temperature and decreasing potentials. These trends correlate with the trends observed for reactivation kinetics but not for H_2 affinity or the kinetics of O_2 attack. Clearly, the rate of recovery is a crucial factor. We present a kinetic and thermodynamic model to account for O_2 tolerance in Re MBH that may be more widely applied to other [NiFe]-hydrogenases.