O2 Reduction to H2O by the multicopper oxidases

Abstract

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...

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

“…Importantly, the large force constant in deoxy Hc indicates that the active site is tightly controlled to keep the Cu-Cu distance short for facile reversible O 2 binding. Alternatively in deoxy Lc T3 , the low force constant allows a large change in Cu-Cu distance with little change in energy, which is required in the reaction coordinate for OOO bond cleavage at the trinuclear Cu cluster in the MCOs (43).…”

How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases

“…An implicit prediction of the model is that O 2 -tolerant respiratory [NiFe]-hydrogenases are high-fidelity four-electron oxidases (Fig. 1), thus extending the subgroup EC.1.X.3 that already includes cytochrome c oxidase (Fe,Cu) (30), blue copper oxidases (Cu) (31), and alternative oxidases (Fe) (32) to include the element Ni as an active component. Compared with the established enzymes, the special oxidase activity of Hyd-1 is low, even under artificially high O 2 concentrations, consistent with it serving to protect the active site, rather than sequestering O 2 from the periplasm.…”

Spectroscopic and computational insight into the activation of O 2 by the mononuclear Cu center in polysaccharide monooxygenases

“…Several classes with either binuclear or trinuclear copper active sites have been identified and their different strategies for O 2 activation have been elucidated (1). These include the multicopper oxidases that use three Cu ions to reduce O 2 to water with very little overpotential (2,3), the coupled binuclear Cu enzymes that are involved in dioxygen transport and monooxygenase reactivity (4), and the noncoupled binuclear Cu monooxygenases that activate O 2 for hydroxylation of peptides and hormones (5). Recently, a class of oxygen activating enzymes with a single copper center has been identified, the polysaccharride monooxygenases [PMOs; often termed lytic polysaccharide monooxygenases (LPMOs), reflecting their ability to break polysaccharides chains and loosen crystalline structure] (6-8), or AA9 to 11 enzymes (AA = auxiliary activity) in the Carbohydrate-Active enZYmes (CAZy) database (9).…”