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 oxidase Fet3p couples four 1e − oxidations of substrate to the 4e − reduction of O 2 to H 2 O. Fet3p uses four Cu atoms to accomplish this reaction: the type 1, type 2, and coupled binuclear type 3 sites. The type 2 and type 3 sites together form a trinuclear Cu cluster (TNC) which is the site of O 2 reduction. This study focuses on mutants of two residues, E487 and D94, which lie in the second coordination sphere of the TNC and defines the role that each plays in the structural integrity of the TNC, its reactivity with O 2 , and in the directional movement of protons during reductive cleavage of the O-O bond. The E487D, E487A, and D94E mutants have been studied in the holo and type 1 depleted (T1D) forms. Residue E487, located near the T3 center, is found to be responsible for donation of a proton during the reductive cleavage of the O-O bond in the peroxide intermediate and an inverse kinetic solvent isotope effect, which indicates that this proton is already transferred when the O-O bond is cleaved. Residue D94, near the T2 site, plays a key role in the reaction of the reduced TNC with O 2 , and drives electron transfer from the T2 Cu to cleave the O-O bond by deprotonating the T2 Cu water ligand. A mechanism is developed where these second sphere residues participate in the proton assisted reductive cleavage of the O-O bond at the TNC.
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 multicopper oxidase Fet3p catalyzes the four-electron reduction of dioxygen to water, coupled to the one-electron oxidation of four equivalents of substrate. To carry out this process the enzyme utilizes four Cu atoms: a type 1, a type 2, and a coupled binuclear, type 3 site. Substrates are oxidized at the T1 Cu, which rapidly transfers electrons, 13 Å away, to a trinuclear copper cluster composed of the T2 and T3 sites where dioxygen is reduced to water in two sequential 2e − steps. This study focuses on two variants of Fet3p, H126Q and H483Q, that perturb the two T3 Cu's, T3α and T3β, respectively. The variants have been isolated in both holo and type 1 depleted (T1D) forms, T1DT3αQ and T1DT3βQ, and their trinuclear copper clusters have been characterized in their oxidized and reduced states. While the variants are only mildly perturbed relative to T1D in the resting oxidized state, in contrast to T1D they are both found to have lost a ligand in their reduced states. Importantly, T1DT3αQ reacts with O 2 but T1DT3βQ does not. Thus loss of a ligand at T3β, but not at T3α, turns off O 2 reactivity, indicating that T3β and T2 are required for the 2e − reduction of O 2 to form the peroxide intermediate (PI), whereas T3α remains reduced. This is supported by the spectroscopic features of PI in T1DT3αQ, which are identical to T1D PI. This selective redox activity of one edge of the trinuclear cluster demonstrates its asymmetry in O 2 reactivity. The structural origin of this asymmetry between the T3α and T3β is discussed as is its contribution to reactivity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.