In nature the four electron reduction of O 2 to H 2 O is carried out by Cytochrome c Oxidase (CcO) and the multicopper oxidases (MCOs). In the former, Cytochrome c provides electrons for pumping protons to produce a gradient for ATP synthesis, while in the MCOs the function is the oxidation of substrates, either organic or metal ions. In the MCOs the reduction of O 2 is carried out at a trinuclear Cu cluster (TNC). Oxygen intermediates have been trapped which exhibit unique spectroscopic features that reflect novel geometric and electronic structures. These intermediates have both intact and cleaved O-O bonds, allowing the reductive cleavage of the O-O bond to be studied in detail both experimentally and computationally. These studies show that the topology of the TNC provides a unique geometric and electronic structure particularly suited to carry out this key reaction in Nature.The multicopper oxidases (MCOs) couple four 1-electron oxidations of substrate to the four electron reductive cleavage of the O-O bond of dioxygen using a minimum of four Cu atoms (table 1). 1,2 Among these four Cu's is a type 1 (T1) or blue Cu site, characterized by an intense S Cys → Cu(II) charge transfer (CT) transition at around 600 nm in the absorption spectrum and a uniquely small A || in its electron paramagnetic resonance (EPR) spectrum. This is the site of substrate oxidation, and from table 1, the MCOs can be divided into two classes depending upon the identity of the substrate. For enzymes such as laccase 3 and ascorbate oxidase, 4 redox active organic molecules which can interact weakly with the enzyme provide the electrons. For MCOs like Fet3p 5 and Ceruloplasmin, 6 the substrate is a metal ion (ferrous in these cases) which binds tightly to a substrate binding site. As shown in figure 1, these substrate binding sites are located near the His ligands of the T1 Cu center. The electron from substrate is first transferred to the T1 and then over >13Å through a Cys-His pathway to a trinuclear Cu cluster (TNC) where O 2 is reduced to water (vide infra). 7 We first consider the electron transfer (ET) pathways to the TNC. ET PathwaysHere we focus on the Fe(II) binding site of the enzyme Fet3p, which is involved in the uptake of iron by yeast. 8 (Studies on this enzyme were performed in collaboration with Prof. Dan Kosman and coworkers.) A variable-temperature, variable-field magnetic circular dichroism (VTVH MCD) methodology we developed in other studies was applied to probe this ferrous site. [9][10][11] From figure 2A dark blue, there is a characteristic feature at 8900 cm −1 in the MCD spectrum corresponding to Fe(II) binding with a high affinity (K B > 10 5 M −1 , from MCD titration studies) to a 6 coordinate site in the protein. 12 In the light blue spectrum this feature is eliminated and a peak at 9700 cm −1 , corresponding to aqueous Fe(II) (green) is observed when Zn(II) is first bound to the substrate site, inhibiting ferroxidase activity.From mutagenesis studies we have found that three carboxylates are involved...

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.

The magnetic and electronic properties of a spin-frustrated ground state of an antiferromagnetically coupled 3-fold symmetric trinuclear copper complex (TrisOH) is investigated using a combination of variable-temperature variable-field magnetic circular dichroism (VTVH MCD) and powder/single-crystal EPR. Direct evidence for a low-lying excited S = (1)/(2) state from the zero-field split ground (2)E state is provided by the nonlinear dependence of the MCD intensity on 1/T and the nesting of the VTVH MCD isotherms. A consistent zero-field splitting (Delta) value of approximately 65 cm(-1) is obtained from both approaches. In addition, the strong angular dependence of the single-crystal EPR spectrum, with effective g-values from 2.32 down to an unprecedented 1.2, requires in-state spin-orbit coupling of the (2)E state via antisymmetric exchange. The observable EPR intensities also require lowering of the symmetry of the trimer structure, likely reflecting a magnetic Jahn-Teller effect. Thus, the Delta of the ground (2)E state is shown to be governed by the competing effects of antisymmetric exchange (G = 36.0 +/- 0.8 cm(-1)) and symmetry lowering (delta = 17.5 +/- 5.0 cm(-1)). G and delta have opposite effects on the spin distribution over the three metal sites where the former tends to delocalize and the latter tends to localize the spin of the S(tot) = (1)/(2) ground state on one metal center. The combined effects lead to partial delocalization, reflected by the observed EPR parallel hyperfine splitting of 74 x 10(-4) cm(-1). The origin of the large G value derives from the efficient superexchange pathway available between the ground d(x2-y2) and excited d(xy) orbitals of adjacent Cu sites, via strong sigma-type bonds with the in-plane p-orbitals of the bridging hydroxy ligands. This study provides significant insight into the orbital origin of the spin Hamiltonian parameters of a spin-frustrated ground state of a trigonal copper cluster.

Laccase is a multicopper oxidase that contains four Cu ions, one type 1 (T1), one type 2 (T2), and a coupled binuclear type 3 Cu pair (T3). The T2 and T3 centers form a trinuclear Cu cluster that is the active site for O2 reduction to H2O. A combination of spectroscopic and DFT studies on a derivative where the T1 Cu has been replaced by a spectroscopically innocent Hg2+ ion has led to a detailed geometric and electronic structure description of the resting trinuclear Cu cluster, complementing crystallographic results. The nature of the T2 Cu ligation has been elucidated; this site is three-coordinate with two histidines and a hydroxide over its functional pH range (stabilized by a large inductive effect, cluster charge, and a hydrogen-bonding network). Both the T2 and T3 Cu centers have open coordination positions oriented toward the center of the cluster. DFT calculations show that the negative protein pocket (four conserved Asp/Glu residues within 12 A) and the dielectric of the protein play important roles in the electrostatic stability and integrity of the highly charged, coordinatively unsaturated trinuclear cupric cluster. These tune the ligand binding properties of the cluster, leading to its high affinity for fluoride and its coordination unsaturation in aqueous media, which play a key role in its O2 reactivity.

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.

Copper cluster sites in biology exhibit unique spectroscopic features reflecting exchange coupling between oxidized Cu's and e − delocalization in mixed valent sites. These novel electronic structures play critical roles in O 2 binding and activation for electrophilic aromatic attack and H atom abstraction, the 4e − /4H + reduction of O 2 to H 2 O, and in the 2e − /2H + reduction of N 2 O. These electronic structure/reactivity correlations are summarized below.Cu proteins play central roles in Fe, Cu, and O 2 metabolism, are related to a range of genetic diseases and are important in biotechnology, detoxification, and the elimination of greenhouse gases. Understanding Cu biochemistry on a molecular level provides mechanisms to improve or inhibit these processes and enhance drug design. The Cu proteins involved in O 2 binding, activation, reduction to H 2 O and the reduction of N 2 O to water and dinitrogen are summarized in Figure 1. The term "coupled" is used here to refer to the antiferromagnetic (AF) "coupling" between paramagnetic metal centers that can lead to a diamagnetic S tot =0 ground state. If two Cu(II)'s, S=1/2, directly overlap they will spin pair. If, however, they are far enough apart so that their d orbitals do not directly overlap but have a bridging ligand this can provide a superexchange pathway (i.e. a delocalized molecular orbital) between the two paramagnetic Cu(II)'s that results in their spin pairing, indirectly through overlap with the bridge. This is described by the exchange Hamiltonian H =−2JS A ·S B which spin couples the two S=1/2's on Cu A and Cu B to form total spins S tot =1 and 0 where for AF coupling the S tot =0 is lower in energy by 2J (J<0).In this paper we will: 1) consider the unique spectral features of the coupled binuclear Cu proteins, hemocyanin (Hc), catechol oxidase, and tyrosinase (Ty), that reflect a novel electronic structure that allows their reversible binding of O 2 (a spin forbidden process) and its activation for electrophilic attack on an aromatic substrate by Ty; 2) contrast this electronic structure to that of the non-coupled binuclear Cu enzymes (i.e. no magnetic interaction between the two Cu(II)'s S=1/2) to evaluate the contribution of these differences in AF exchange coupling to the reaction mechanisms, where the non-coupled binuclear Cu sites in dopamine α-monooxygenase (DβM) and peptidylglycine α-hydroxylating monooxygenase (PHM) activate O 2 for H-atom abstraction; 3) Extend these studies to the trinuclear Cu cluster site in the multicopper oxidases, where the exchange coupling among the three coppers plays a central role in the 4e − /4H + reduction of O 2 to H 2 O; and 4) Consider how the interactions among the coppers in the μ 4 sulfide bridged tetranuclear Cu z cluster promote the 2e − /2H + cleavage of the N-O bond by N 2 O reductase. for trigonal bipyramidal geometries. Peroxide has a doubly degenerate highest occupied molecular orbital (HOMO) set which will split in energy upon binding to Cu(II), the π* σ being stabilized to deeper bi...

The ground state electronic and magnetic properties of one of the possible structures of the trinuclear Cu II site in the native intermediate (NI) of the multicopper oxidases, the μ 3 -oxo bridged structure, are evaluated using the C 3 -symmetric Cu 3 II complex, μ 3 O. μ 3 O is unique in that no ligand, other than the oxo, contributes to the exchange coupling. However, μ 3 O has a ferromagnetic ground state, inconsistent with that of NI. Therefore, two perturbations have been considered: protonation of the μ 3 -oxo ligand and relaxation of the μ 3 -oxo ligand into the Cu 3 plane. Notably, when the oxo-ligand is sufficiently close to the Cu 3 plane (< 0.3 Å), the ground state of μ 3 O becomes antiferromagnetic and can be correlated to that of NI. In addition, the ferromagnetic 4 A ground state of μ 3 O is found from variable-temperature EPR to undergo a zero-field splitting (ZFS) of 2D = -5.0 cm -1 , which derives from the second-order anisotropic exchange. This allows evaluation of the σ-to-π excited state exchange pathways and provides experimental evidence that the orbitally-degenerate 2 E ground state of the antiferromagnetic μ 3 O would also undergo a ZFS by the first-order antisymmetric exchange that has the same physical origin as the anisotropic exchange. The important contribution of the μ 3 -oxo bridge to the ground-to-ground and ground-to-excited state superexchange pathways that are responsible for the isotropic, antisymmetric and anisotropic exchange are discussed.

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