Mechanistic studies of the multicopper oxidases [electronic resource] : reduction of the native intermediate and molecular origin of rapid intramolecular electron transfer in turnover

The Multicopper Oxidases (MCOs) are the family of enzymes that couple the four-electron reduction of dioxygen to water with four 1-electron oxidations of substrates. These enzymes contain a Type 1 (T1) or blue Cu center that is solvent accessible and where substrate is oxidized, reducing this Cu. These electrons are then transferred over a distance of ~13 Å through the protein via a Cys-His pathway to a trinuclear cluster (TNC), composed of a Type 3 Cu pair and a Type 2 Cu, where dioxygen binds and is reduced to water. The mechanism of dioxygen reduction to water by the MCOs is well-characterized and proceeds in two, two-electron steps. The fully reduced enzyme reacts with dioxygen to form the Peroxy Intermediate, where dioxygen is bound as peroxide to the TNC. The O-O bond is cleaved at this point to produce the native intermediate (NI), which is fully oxidized with all three Cu's of the TNC bridged by a central oxo and the T3s are additionally bridged by a hydroxo. Both moieties originate from the 4-electron reduction of dioxygen. This contrasts the resting state where the only bridging ligand is a T3 hydroxo. A long-standing problem concerning the catalytic mechanism of the MCOs was the process by which these enzymes are rereduced in the catalytic cycle. Specifically, Rhus vernicifera Laccase exhibits a turnover rate of 560 s-1 while the intramolecular electron transfer (IET) from the T1 to the TNC of the resting state of this enzyme has been measured to be 1.1 s-1 and therefore not consistent with turnover. We have now experimentally determined that the reduction of NI by ET from the T1 is fast relative to its decay and obtained an IET rate lower-limit of > 700 s-1 for the first electron reduction. Thus proves that NI is the catalytically relevant fully oxidized form of the MCOs; not the fully oxidized resting formed as studied crystallographically. Thus, the first IET rate in NI reduction is more than three orders of magnitude faster than the IET rate in the resting oxidized state. Computations show that this rate difference derives from a larger driving force for proton-coupled electron transfer in NI compared to the resting state due to the strong basicity of the central oxo of NI, where the resting TNC site lacks a strongly basic ligand. In addition to the first IET, there are two remaining IET steps in the reduction of NI to the fully reduced state to complete the catalytic cycle. Kinetic analysis shows that the second IET step is reversible (K ≈ 1), the third is irreversible and both are fast with lower-limits of > 500 s-1. A signal is observed in freeze quench EPR that corresponds to the 1 electron hole intermediate (two electron reduced NI). Kinetic and spectroscopic results coupled to DFT calculations reveal the mechanism of the 3 electron / 3 proton reduction of NI, where all three catalytically relevant intramolecular electron transfer (IET) steps are rapid and involve three different structural changes. The first IET process is a concerted electron and proton transfer (EPT) process made rapid due to the driving force supplied by the protonation of the basic central oxo of NI. The Second IET has a low driving force, but also a low reorganization energy due to a proton transfer / electron transfer stepwise process. The third IET is a concerted EPT process but in this case driven by the extrusion of product waters from the fully reduced TNC. These three rapid IET processes reflect the sophisticated mechanistic flexibility of the TNC to enable rapid turnover. Importantly, all three of these IET steps are rapid because of the basicity of the dioxygen-derived ligands that arise from O-O bond cleavage. In catalysis, the TNC performs the four-electron reduction of dioxygen to the water level in the formation of NI, but only after NI is fully reduced are the water products of dioxygen reduction fully formed and extruded from the cluster to enable reduction of another equivalent of dioxygen. This defines a unifying catalytic mechanism for the MCOs where O-O bond cleavage and three rapid IETs are coupled to enable fast turnover in oxidation catalysis.

Keywords: Chemistry.
Advisor: Solomon Edward I. primary advisor. Thesis advisor
Hodgson Keith
Waymouth Robert M. advisor. Thesis advisor