A Novel Mixed Valence Form of Rhus vernicifera Laccase and Its Reaction with Dioxygen to Give a Peroxide Intermediate Bound to the Trinuclear Center

Abstract

Multicopper oxidase belongs to the class of enzyme containing four or more Cu centers in a protein molecule (1). The minimum functional unit of multicopper oxidase comprises a set of one type 1 Cu, one type 2 Cu, and a pair of type 3 Cus. The type 1 Cu functions as the electron mediator from a substrate to the trinuclear center composed of the type 2 Cu and type 3 Cus, where dioxygen is bound and reduced to two water molecules. All these copper sites are indispensable for the four-electron reduction of dioxygen without the release of activated oxygen species as intermediates.When lacquer tree laccase, the best studied multicopper oxidase, was reduced fully and reacted with dioxygen, the three-electron reduced form of dioxygen appeared within 15 ms with a life-time of s to min order depending on the pH (2, 3). We spectroscopically and magnetically characterized this species as the oxygen-centered racial bound to the trinclear copper center, finding that it decayed on accepting the final electron from type 2 Cu under the control of proton transfer gating (4). In order to detect the preceding inter-1 This study was supported by Grants-in-Aid for Scientific Research, C 11640558 and B 13440194, from the Ministry of Education, Science, Sports and Culture of Japan, and the Joint Studies Program of the Institute for Molecular Science 1 To whom correspondence should be addressed. E-mail: tsO513@ kenroku.kanazawa-u.ac.jp Abbreviations: TloxT23red, type 1 Cu as Cu(II), and type 2 and 3 Cus as Cud); TloxT3red, type 2 Cu-depleted laccase in which type 1 Cu is Cu(II) and type 3 Cus are reduced; T12oxT3red, type 1 and 2 Cus as Cu(ll), and type 3 Cus as Cu(D; EPR, electron paramagnetic resonance; CD, circular dichroism; T, tesla (1 T = 10,000 G); TlHg, mercury derivative of laccase at the type 1 Cu site; FTTR, Fouriertransform infrared; DPPH, diphenylpicrylhydrazyl.© 2001 by The Japanese Biochemical Society. mediate, such as the two-electron reduced form of dioxygen, we investigated the earlier reaction process of the reduced laccase with dioxygen by means of stopped-flow spectroscopy (2). However, it was impossible to detect the two-electron reduced form of dioxygen because the diffusion of dioxygen towards the active site was too slow (ms order) compared with the rate of the electron transfer from type 1 Cu to the putative peroxide intermediate (the two-electron reduced form) (|xs or less order). The content of the two-electron reduced species has been estimated to be a few percent and only the formation of the metastable three-electron reduced form of dioxygen (the radical intermediate) could be observed insofar as the reactions of the native enzyme were studied (3).The type 2 Cu in laccase has been selectively depleted through the action of potassium hexacyanoferratefll) CK,-[Fe(CN) 6 ]) on the resting enzyme under anaerobic conditions, followed by the action of dimethylglyoxime and EDTA (5). In the type 2 Cu-depelted laccase the type 1 Cu is cupric and the coupled type 3 Cus are cuprous (TloxT3red, which has been simply ab...

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

“…These spectral features are very similar to those produced by a mercury-substituted or mixed valent laccase (13,14) and Cys-to-Ser mutants of bilirubin oxidase (15) and Fet3p (16), indicating that multicopper oxidases commonly pass through intermediate I. The intensity of intermediate I band was much higher at 340 nm (⑀ ϭ 9,000) than at 315-330 nm due to the resting trinuclear copper center (⑀ ϭ 4,500 -5,300) (Figs.…”

“…Two reaction intermediates have been detected during reactions of some multicopper oxidases. One of them, intermediate I, could be trapped by the following modified multicopper oxidases so as to interrupt the electron transfer from the type I copper: a plant laccase whose type I copper was substituted with mercury (13); a mixed valent laccase in which the type I copper was oxidized, but the trinuclear copper center was reduced (14); and a Cys 3 Ser mutant of bilirubin oxidase (15) and Fet3p (16) whose type I copper center became vacant. Although the trinuclear copper center must be fully reduced to produce intermediate I, it has been considered to be a twoelectron reduced form and, therefore, also called the peroxide intermediate (13,16).…”

“…Although the trinuclear copper center must be fully reduced to produce intermediate I, it has been considered to be a twoelectron reduced form and, therefore, also called the peroxide intermediate (13,16). Another reaction intermediate, II, also called the native intermediate, has been detected at the final stage of a single turnover (15,(17)(18)(19) (13)(14)(15)(16)(17)(18)(19).…”

Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase

“…The His456.458Lys mutant showed BO and ferroxidase activities, indicating that the electron from a substrate is finally transferred to dioxygen via type I Cu and the mutated trinuclear center (18)(19)(20)(21). However, the drastic decreases in the enzyme activities of His456.458Lys would have been brought about by the change in the redox potential of the type III Cu centers (the substitution of Lys for His would lower the redox potential of type III Cu, significantly suppressing the rate of intramolecular electron transfer) and/or by lowering of the affinity of the trinuclear center towards dioxygen (Table 2).…”

An O‐Centered Structure of the Trinuclear Copper Center in the Cys500Ser/Glu506Gln Mutant of CueO and Structural Changes in Low to High X‐Ray Dose Conditions

“…Intermediates I can be trapped during the course of reactions of the MCO derivatives such as: the Hg-substituted laccase at the T1Cu center, [7] the mixed valent laccase with T1Cu in the cupric state and T2Cu and T3Cus in the cuprous state, [8] CueO, [9] bilirubin oxidase, [10] and Fet3p, [11] in which the T1Cu site is vacant as a result of the replacement of the Cys ligand with Ser. Since the intermediate I does not have an electron paramagnetic resonance (EPR) signal ( Figure S1 in Supporting Information), [7][8][9][10][11] it has been proposed that a peroxide species is bound to the TNC in a -1,2-peroxofashion, and accordingly, one of the three copper ions remains in the cuprous state. However, direct evidence to support the presence of peroxide or the oxidation state of the copper centers in the TNC has not been published.…”