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

Abstract

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 O2 to H2O. Reaction of the fully reduced enzyme with O2 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 NI 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 O2 at the trinuclear Cu site. Correlation of PI to NI 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 NI and the involvement of the simultaneous two-electron-transfer process. A low activation barrier (calculated approximately 5-6 kcal/mol and experimental approximately 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 O22- sigma* 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.

O2 Reduction to H2O by the multicopper oxidases

“…Importantly, if an Asp, present in the crystal structure (D94 in Fet3p) near the T2 Cu, is included in the calculation a structure consistent with the spectrum is obtained with the peroxide binding η 2 to the T3 Cu B (nearer to the Asp) and η 1 to each of the other two Cu's (figure 13, right). 38 This computational result is consistent with experimental results where the D94A and D94N mutants do not react with O 2 , while replacing D with a negatively charged E residue allows the O 2 reaction to occur. 39,40 This μ 3 -1,1,2 structure appears to be required for the irreversible binding of O 2 and its activation for further reduction.…”

“…From electronic structure calculations supported by spectroscopic data (figure 14) both the T2 and T3Cu B are oxidized due to the presence of the Asp which reduces their potential, and each has significant π* σ character leading to the strong AF coupling determined experimentally for PI. 38 …”

“…Together with the large driving force, these FMOs lead to the very low barrier for the two electron reductive cleavage of the peroxide O-O bond. 38 …”

“…Note that T2 and T3A transfer two electrons into the peroxide σ* orbital and T3B is oxidized and acts as a Lewis Acid, similar to a proton, in stabilizing the σ* orbital. 38 …”

Geometric and electronic structure differences between the type 3 copper sites of the multicopper oxidases and hemocyanin/tyrosinase

“…Alternatively, the deoxy Lc T3 Cu site in the multicopper oxidases is electrostatically stable and does not react with O 2 when the T2 Cu is not present. When the type 2 Cu is also present, the trinuclear Cu cluster does react with O 2 to form a 2-electron reduced peroxide, bridging between the type 2 and type 3 Cu centers (27,28). The trinuclear Cu cluster further promotes cleavage of the OOO bond by stabilizing the products of peroxide reduction (oxo and hydroxo moieties) through multiple bridging interactions requiring a large change in Cu-Cu distance.…”