Reversible [4Fe-3S] cluster morphing in an O2-tolerant [NiFe] hydrogenase

Hydrogenases catalyze the reversible oxidation of H-2 into protons and electrons and are usually readily inactivated by O-2. However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O-2 that enables their host organisms to utilize H-2 as an energy source at high O-2. This feature is crucially based on a unique six cysteine-coordinated [4Fe-3S] cluster located close to the catalytic center, whose properties were investigated in this study using a multidisciplinary approach. The [4Fe-3S] cluster undergoes redox-dependent reversible transformations, namely iron swapping between a sulfide and a peptide amide N. Moreover, our investigations unraveled the redox-dependent and reversible occurence of an oxygen ligand located at a different iron. This ligand is hydrogen bonded to a conserved histidine that is essential for H-2 oxidation at high O-2. We propose that these transformations, reminiscent of those of the P-cluster of nitrogenase, enable the consecutive transfer of two electrons within a physiological potential range.