Dynamic Proton Sharing and Second-Sphere Gating Control Catalytic States in [NiFe] Hydrogenase

[NiFe] hydrogenases catalyze reversible oxidation of molecular hydrogen (H₂) through four redox states (Ni-SI_a, Ni-L, Ni-C, Ni-R), yet the spectroscopic heterogeneity of the Ni-L and Ni-R intermediates has long obscured their mechanistic roles. Using multiscale quantum mechanics/molecular mechanics (QM/MM) calculations, long-time scale molecular dynamics (MD), quantum cluster models, and electron paramagnetic resonance (EPR) and infrared spectroscopy (IR) spectroscopy simulations, we resolve the structural and electronic origins of this heterogeneity. We show that the spectroscopic heterogeneity of Ni-L and Ni-R arises from Cys546-protonated and Glu34-protonated configurations (residue numbering based on Desulfovibrio vulgaris Miyazaki F, referred as DvMF hereafter) along the proton-transfer coordinate, thermodynamically favoring the Glu34-protonated form. A conserved Cys546-Glu34 proton-sharing motif is stabilized by conformationally flexible second-sphere residues Thr18 and Arg479, which strongly modulate catalytic-state energetics and govern proton localization in both Ni-L and Ni-R. Only Glu34-protonated models quantitatively reproduce experimental EPR g-tensors and hyperfine couplings constants (HFC), while Cys546 deprotonation yields characteristic CO/CN red shifts. These results, established for the O₂-sensitive DvMF and further supported by analogous protonation energetics in the O₂-tolerant Escherichia coli Hyd-1, suggest that the Cys-Glu proton-sharing motif and its second-sphere gating mechanism may be broadly conserved within Group 1 [NiFe] hydrogenases. These findings reconcile long-standing models of Ni-L/Ni-R heterogeneity and reveal how second-sphere dynamics gate proton transfer in [NiFe] hydrogenase, offering design principles for next-generation bioinspired hydrogen-evolution catalysts.