Electronic Structure of the Peroxy Intermediate and Its Correlation to the Native Intermediate in the Multicopper Oxidases: Insights into the Reductive Cleavage of the O-O Bond

摘要

The multicopper oxidases (MCOs) utilize a blue type 1 (T1) copper site and a trinuclear Cu cluster composed of a type 2 (T2) and a binuclear type 3 (T3) site that together catalyze the four-electron reduction of O_2 to H_2O. Reaction of the fully reduced enzyme with O_2 proceeds via two sequential two-electron steps generating the peroxy intermediate (PI) and the native intermediate (NI). While a detailed description of the geometric and electronic structure of Nl has been developed, this has been more elusive for PI largely due to the diamagnetic nature of its ground state. Density functional theory (DFT) calculations have been used to correlate to spectroscopic data to generate a description of the geometric and electronic structure of PI. A highly conserved carboxylate residue near the T2 site is found to play a critical role in stabilizing the PI structure, which induces oxidation of the T2 and one T3 Cu center and strong superexchange stabilization via the peroxide bridge, allowing irreversible binding of O_2 at the trinuclear Cu site. Correlation of PI to Nl is achieved using a two-dimensional potential energy surface generated to describe the catalytic two-electron reduction of the peroxide O-O bond by the MCOs. It is found that the reaction is thermodynamically driven by the relative stability of Nl and the involvement of the simultaneous two-electron-transfer process. A low activation barrier (calculated ～5-6 kcal/mol and experimental ～3-5 kcal/mol) is produced by the triangular topology of the trinuclear Cu cluster site, as this symmetry provides good donor-acceptor frontier molecular orbital (FMO) overlap. Finally, the O-O bond cleavage in the trinuclear Cu cluster can be achieved via either a proton-assisted or a proton-unassisted process, allowing the MCOs to function over a wide range of pH. It is found that while the proton helps to stabilize the acceptor O_2~(2-) σ~* orbital in the proton-assisted process for better donor-acceptor FMO overlap, the third oxidized Cu center in the trinuclear site assumes the role as a Lewis acid in the proton-unassisted process for similarly efficient O-O bond cleavage.