CO dehydrogenase (CODH) is an environmentally crucial bacterial enzyme that oxidizes CO to CO2 at a Mo-Cu active site. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the protonation state of the water-derived equatorial ligand coordinated at the Mo-center, as well as the nature of intermediates formed during the catalytic cycle. To address the protonation state of the equatorial ligand, we have developed a realistic in silico QM model (~179 atoms) containing structurally essential residues surrounding the active site. Using our QM model, we examined each plausible combination of redox states (Mo(VI)-Cu(I), Mo(V)-Cu(II), Mo(V)-Cu(I), and Mo(IV)-Cu(I)) and Mo-coordinated equatorial ligands (O(2-), OH(-), H2O), as well as the effects of second-sphere residues surrounding the active site. Herein, we present a refined computational model for the Mo(VI) state in which Glu763 acts as an active site base, leading to a MoO2-like core and a protonated Glu763. Calculated structural and spectroscopic data (hyperfine couplings) are in support of a MoO2-like core in agreement with XRD data. The calculated two-electron reduction potential (E = -467 mV vs. SHE) is in reasonable agreement with the experimental value (E = -558 mV vs. SHE) for the redox couple comprising an equatorial oxo ligand and protonated Glu763 in the Mo(VI)-Cu(I) state and an equatorial water in the Mo(IV)-Cu(I) state. We also suggest a potential role of second-sphere residues (e.g., Glu763, Phe390) based on geometric changes observed upon exclusion of these residues in the most plausible oxidized states.
Keywords: CO dehydrogenase; Computational model; Density functional theory; Molybdenum–copper bimetallic site; Quantum mechanics.