A realistic in silico model for structure/function studies of molybdenum–copper CO dehydrogenase

CO dehydrogenase (CODH) is an environmentally crucial bacterial enzyme that oxidizes CO to CO 2 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 − , H 2 O), 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 MoO 2 -like core and a protonated Glu763. Calculated structural and spectroscopic data (hyperfine couplings) are in support of a MoO 2 -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.