Four-electron Reduction of Dioxygen by a Multicopper Oxidase, CueO, and Roles of Asp112 and Glu506 Located Adjacent to the Trinuclear Copper Center

Kunishige Kataoka,Ryosuke Sugiyama,Shun Hirota,Megumi Inoue,Kanae Urata,Yoichi Minagawa,Daisuke Seo,Takeshi Sakurai

doi:10.1074/jbc.m808468200

Kunishige Kataoka, Ryosuke Sugiyama + Show 6 more

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https://doi.org/10.1074/jbc.m808468200

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Abstract

The mechanism of the four-electron reduction of dioxygen by a multicopper oxidase, CueO, was studied based on reactions of single and double mutants with Cys(500), a type I copper ligand, and the noncoordinating Asp(112) and Glu(506), which form hydrogen bonds with the trinuclear copper center directly and indirectly via a water molecule. The reaction of C500S containing a vacant type I copper center produced intermediate I in an EPR-silent peroxide-bound form. The formation of intermediate I from C500S/D112N was restricted due to a reduction in the affinity of the trinuclear copper center for dioxygen. The state of intermediate I was realized to be the resting form of C500S/E506Q and C500S of the truncated mutant Deltaalpha5-7CueO, in which the 50 amino acids covering the substrate-binding site were removed. Reactions of the recombinant CueO and E506Q afforded intermediate II, a fully oxidized form different from the resting one, with a very broad EPR signal, g < 2, detectable only at cryogenic temperatures and unsaturated with high power microwaves. The lifetime of intermediate II was prolonged by the mutation at Glu(506) involved in the donation of protons. The structure of intermediates I and II and the mechanism of the four-electron reduction of dioxygen driven by Asp(112) and Glu(506) are discussed.

Highlights

CueO has the same catalytic copper centers as other multicopper oxidases: a type I copper that mediates electron transfer and a trinuclear copper center comprised of a type II copper and a pair of type III copper atoms, where dioxygen is reduced to two water molecules [5, 7]
In a preliminary study on the Asp112 mutants [21], we showed that this acidic amino acid functions in the binding of dioxygen at the trinuclear copper center and may be involved in the donation of protons to the reaction intermediate(s)
The d-d transition band due to the type II and III copper atoms, which had been masked by the intense d-d band of the type I copper at ϳ750 nm (⑀ ϭ 2,000) in recombinant form of CueO (rCueO), became observable at ϳ710 nm (⑀ ϭ ϳ500)

Summary

Introduction

CueO has the same catalytic copper centers as other multicopper oxidases: a type I copper that mediates electron transfer and a trinuclear copper center comprised of a type II copper and a pair of type III copper atoms, where dioxygen is reduced to two water molecules [5, 7]. Mediates have a half-life in the order of seconds to minutes, but information to directly show their structures has not been obtained yet They afford analogous absorption bands at ϳ330 –350, 450 – 470, and 680 nm, of which the former two bands have been assigned to the charge transfer from a certain oxygen group to Cu2ϩ (␴ and ␲ transitions) and the latter to the d-d transitions of the trinuclear copper center in the cupric state. The d-d transitions of intermediate II are masked by strong absorption due to the oxidized type I copper [13,14,15,16,17,18,19]

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