Electrochemical investigations of the Membrane-Bound [NiFe] Hydrogenase of Ralstonia eutropha

Hydrogenases are structurally complex enzymes that catalyse the production and oxidation of H2 in a wide variety of microorganisms. The catalytic properties of these enzymes are related to the interaction between their redox-active metal cofactors. O2-tolerant [NiFe] hydrogenases catalyse H2 oxidation in the presence of O2, which normally completely inhibits other hydrogenases. Their (in)activation mechanism is of fundamental importance for H2-based energy technologies.
The present study has characterized the catalytic properties of the full heterotrimeric membrane bound hydrogenase (MBH) in native-like conditions by using a novel approach for immobilizing the enzyme onto the electrode. With the use of the tethered bilayer lipid membrane (tBLM) approach, the study obtained mechanistic insights relevant to the in vivo functioning of the enzyme. The MBH, inserted into the tethered lipid membrane, in equilibrium with the quinone pool, was probed in cyclic voltammetry and chronoamperometry experiments.
The catalytic properties displayed at oxidizing potentials revealed that the heterotrimeric MBH undergoes anaerobic oxidative inactivation to a much smaller extent compared to the heterodimeric sub-complex, which was probed in previous protein film electrochemistry studies. In addition, the enzyme recovers after aerobic inactivation under oxidizing conditions without the application of reducing potentials.
The reactivation kinetics of MBHwt and that of an MBH variant with the metal cofactor configuration of an O2-sensitive [NiFe] hydrogenase were probed under oxidative substrate-limiting conditions. The results show that the O2 sensitive mutant reactivates faster than MBHwt. This indicates that protection against oxidative damage is achieved by tuning electron transfer to the active site with the scope of preventing the formation of reactive species that would lead to irreversible inactivation.