A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases

Abstract

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 "O(2)-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.

Fig. 1.

A simplified scheme showing the reactions of standard [NiFe]-hydrogenases. Active hydrogenase molecules catalyze H₂/H⁺cycling, and are subject to aerobic inactivation, forming either the Ready or Unready forms, depending on the availability of electrons and protons. The Ready form can also be formed anaerobically at oxidizing potentials. (Adapted from ref. with permission from the Royal Society of Chemistry.)

Fig. 2.

A typical experiment to determine the rate of O₂ reaction with Re MBH. (A) The current vs. time trace (black) with the fit to film loss/anaerobic inactivation (gray). (B) The corrected data (black), with the fit to the initial attack by O₂ (gray). Experimental conditions were pH 5.5, 10° C, +0.192 V, electrode rotation rate = 4,500 rpm.

Fig. 3.

Reactivation of aerobically and anaerobically inactivated states of Re MBH. (A and B) Typical experiments to measure the rate of reactivation of Re MBH under 100% H₂ following anaerobic (A) and aerobic (B) inactivation. In these examples, reactivation was measured at +0.192 V. (C) Rates of reactivation as a function of potential with a fit to the data overlaid. Data for the reactivation of Av MBH from the Unready state (filled squares, taken from reference 16, pH 6, 45° C) are also shown, and have been multiplied by 10 for clarity (unfilled squares). Sigmoidal fits are also shown. (D) Voltammograms (100% H₂, 0.1 mV s⁻¹) of Re MBH (bold) and Av MBH (dashed), showing recovery after aerobic inactivation at 392 mV under N₂. All at pH 5.5, 10° C, electrode rotation rate = 2,500 rpm.

Fig. 4.

The kinetic scheme used to model O₂ tolerance. Re MBH is abbreviated as “E” and is shown in the reduced active form (E_act), as an adduct with H₂ or O₂ (E_act−H₂ and E_act−O₂, respectively) or in the Ready (E_Ready) or Unready (E_Unready) state.

Fig. 5.

Simulations obtained by applying Eq. 1 to Re MBH. (A) Limiting H₂ oxidation currents at varying O₂ concentrations, determined experimentally (black) and calculated from the model (gray). (B–D) show the effect of altering the percentage O₂ (B), the electrode potential (C), and k_A (D) on the catalytic currents calculated from the model. All conditions are pH 5.5, 10° C. B and D are at 0.122 V, and C and D are at 10% O₂.