Variable-Temperature, Variable-Field Magnetic Circular Dichroism Studies of Tris-Hydroxy- and μ₃-Oxo-Bridged Trinuclear Cu(II) Complexes:  Evaluation of Proposed Structures of the Native Intermediate of the Multicopper Oxidases

Abstract

Multicopper oxidases catalyze the 4e- reduction of O2 to H2O. Reaction of the fully reduced enzyme with O2 produces the native intermediate (NI) that consists of four oxidized Cu centers, three of which form a trinuclear cluster site, all bridged by the product of full O2 reduction. The most characteristic feature of NI is the intense magnetic circular dichroism pseudo-A feature (a pair of temperature-dependent C-terms with opposite signs) associated with O --> Cu(II) ligand-to-metal charge transfer (LMCT) that derives from the strong Cu-O bonds in the trinuclear site. In this study, the two most plausible Cu-O structures of the trinuclear site, the tris-mu2-hydroxy-bridged and the mu3-oxo-bridged structures, are evaluated through spectroscopic and electronic structure studies on relevant model complexes, TrisOH and mu3O. It is found that the two components of a pseudo-A-term for TrisOH are associated with LMCT to the same Cu that are coupled by a metal-centered excited-state spin-orbit coupling (SOC), whereas for mu3O they are associated with LMCT to different Cu centers that are coupled by oxo-centered excited state SOC. Based on this analysis of the two candidate models, only the mu3-oxo-bridged structure is consistent with the spectroscopic properties of NI. The Cu-O sigma-bonds in the mu3-oxo-bridged structure would provide the thermodynamic driving force for the 4e- reduction of O2 and would allow the facile electron transfer to all Cu centers in the trinuclear cluster that is consistent with its involvement in the catalytic cycle.

O2 Reduction to H2O by the multicopper oxidases

“…For the μ 3 -oxo structure in figure 21B, this involves oxo CT transitions to two different Cu centers which SOC on the oxygen (p x ↔ p y ). 44 The phases of this mechanism are consistent with the negative component of the pseudo-A term being higher in energy, as observed in the μ 3 -oxo model ( figure 20B) and NI ( figure 20C). From the temperature dependence of the MCD data on NI, 41 the components of its pseudo-A term involve CT transitions to different Cu centers (see labels in figure 20C).…”

O2 and N2O Activation by Bi-, Tri-, and Tetranuclear Cu Clusters in Biology

“…Model complexes exist with both structures and both exhibit MCD pseudo-A terms (of opposite sign) associated with the hydroxo or oxo to Cu(II) CT transitions of the trinuclear Cu(II) cluster (Figure 14A bottom). 34 The mechanism for pseudo-A terms requires two perpendicular CT transitions being spinorbit coupled in a third direction at one center. For the tris OH − system, this would involve CT transitions from two OH − ligands to one Cu(II) center which provides the spin-orbit coupling.…”

The two oxidized forms of the trinuclear Cu cluster in the multicopper oxidases and mechanism for the decay of the native intermediate

“…A combination of Cu K-edge x-ray spectroscopy (XAS) and magnetic circular dichroism (MCD) has unambiguously demonstrated that NI is a fully oxidized form with fully reduced O 2 (3). Recent model studies combined with calculations have further demonstrated that the three Cu(II) centers in the trinuclear site are all bridged by a 3 -oxo ligand (8,9). In the absence of reducing substrate, NI slowly decays to the resting enzyme, in which the one remaining O atom of the O 2 is terminally bound as OH Ϫ to the T2 site, as indicated by 18 O isotope ratio MS (IRMS) (10,11) and 17 O EPR (12) experiments.…”