Bioelectrochemical analysis of thermodynamics of the catalytic cycle and kinetics of the oxidative inactivation of oxygen-tolerant [NiFe]-hydrogenase

• [NiFe]-hydrogenase allows direct electron transfer-type bioelectrocatalysis.
• The enzyme redox state is reversible to give Nernstian response in the catalytic wave.
• Catalytic response has thermodynamically been analyzed on two-step one-electron transfer model.
• The oxidative inactivation has been kinetically analyzed using the catalytic current as a measure.
• The catalytic wave has been successfully simulated with the evaluated parameters.

Membrane-bound [NiFe]-hydrogenase from Hydrogenovibrio marinus (HmMBH) is an O2-tolerant enzyme and allows direct electron transfer (DET)-type bioelectrocatalysis for the H2 oxidation. Very fast interfacial electron transfer occurs between the [NiFe]-active site of HmMBH and the electrode, and the potential dependence of the steady-state DET-type catalytic current has been analyzed on a thermodynamic model of a two-step one-electron transfer to get a Pourbaix diagram of the catalytic center. A reversible and oxidative inactivation that occurs when the [NiFe]-hydrogenases are suffering from the oxidative stress at high electrode potentials or high solution potentials has been kinetically analyzed for the time-dependence of the steady-state catalytic current as a measure. The kinetic analysis has shown that the rate-determining step of the oxidative inactivation is not electrochemical but chemical process and that the rate of the reductive reactivation is determined by the electrochemical process. The observed catalytic waves, especially the dependence of the waves on the scan rate and the hydrogen concentration, have been well reproduced by simulation with the thermodynamic and kinetic parameters evaluated here.

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