Fully Oxidized State of the Oxygen-Tolerant [NiFe] Hydrogenase from <i>Hydrogenophilus thermoluteolus</i> SH: A Quantum Mechanics Cluster and Quantum Mechanics/Molecular Mechanics Study

The oxygen tolerance of some [NiFe] hydrogenase enzymes is crucial for designing efficient bioinspired catalysts for sustainable hydrogen production and advancing renewable energy technologies. To investigate this, we employed a quantum mechanical (QM) cluster model and quantum mechanics/molecular mechanics (QM/MM) calculations to study the fully oxidized state of the [NiFe]-hydrogenase from <i>Hydrogenophilus thermoluteolus</i> SH. Our analysis focused on the structural and electronic properties of the enzyme's active site across different spin states, including closed-shell singlet (CS, S = 0), high-spin triplet (HS, S = 1), and open-shell singlet broken symmetry (BS, S = 0). Using a comprehensive structural model (>300 atoms), we identified the ground state of the fully oxidized enzyme state to be a spin-coupled BS Ni(III)Fe(III) oxidation state, where residues beyond the first coordination sphere primarily contribute sterically. Notably, natural bond order calculations revealed an unusual three-center two-electron bond at the active site, which may enhance the open-shell ground state stability and the enzyme's resilience under oxidative conditions. Our comparative study of QM and QM/MM methods provides insights into their performance, facilitating and guiding the choice of suitable enzyme models when studying other metalloproteins.