Spectroscopic and Kinetic Studies on the Oxygen-centered Radical Formed during the Four-electron Reduction Process of Dioxygen byRhus vernicifera Laccase

Huang, Hongwei; Zoppellaro, Giorgio; Sakurai, Takeshi

doi:10.1074/jbc.274.46.32718

Cited by 64 publications

(94 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When dioxygen accepts two-electrons in the antibonding orbital, the bond between two oxygen atoms becomes single. Once another electron is supplied to the vacant antibonding orbital, the bond-order becomes 0.5, the O-O bond being cleaved formally to give O 2 " (frLft) and 0~\ (oxyl and/or hydroxyl radical), as we have recently characterized in detail (4). It is considered that a similar reaction pathway is followed by all multicopper oxidases such as ceruloplasmin, ascorbate oxidase, bilirubin oxidase, etc.…”

Section: Resultsmentioning

confidence: 96%

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

Zoppellaro¹,

Sakurai²,

Huang³

2001

Journal of Biochemistry

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 96%

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

Zoppellaro¹,

Sakurai²,

Huang³

2001

Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…When the Met ligand of the type I copper in bilirubin oxidase was replaced with Gln, the redox potential of type I copper shifted negatively by ca. The electron transfer processes of MCOs and NIR from the substrate to type I copper and from type I copper to the catalytic center have been studied with the stopped-flow technique, flash-photolysis, and pulse-radiolysis [37,51,52, [97][98][99][100][101][102][103][104][105][106]. The initial electron transfer process from the substrate to type I copper involves the association of substrate with the protein molecule, and accordingly, the proper selection of the substrate is extremely important to study the initial electron transfer process.…”

Section: Function Of Type I Copper In Multicopper Oxidasesmentioning

confidence: 99%

“…The initial electron transfer process from the substrate to type I copper involves the association of substrate with the protein molecule, and accordingly, the proper selection of the substrate is extremely important to study the initial electron transfer process. Unlike the initial stage of the MCO reaction, the succeeding intramolecular electron transfer pathway from type I copper to the trinuclear copper center and the four-electron reduction of dioxygen [56, 102,103] are not affected by how the reaction is started, although these processes will be accelerated under steady state conditions in the presence of excess substrate [101].…”

Section: Function Of Type I Copper In Multicopper Oxidasesmentioning

confidence: 99%

Structure and function of type I copper in multicopper oxidases

Sakurai

Kataoka

2007

Cell. Mol. Life Sci.

128

View full text Add to dashboard Cite

Abstract. The type I copper center in multicopper oxidases is constructed from 1Cys2His and weakly coordinating 1Met or the non-coordinating 1Phe/1Leu, and it exhibits spectral properties and an alkaline transition similar to those of the blue copper center in blue copper proteins. Since the type I copper center in multicopper oxidases is deeply buried inside the protein molecule, electron transfers to and from type I copper are performed through specific pathways: the hydrogen bond between an amino acid located at the substrate binding site and a His residue coordinating type I copper, and the His-Cys-His sequence connecting the type I copper center and the trinuclear copper center comprised of a type II copper and a pair of type III coppers. The intramolecular electron transfer rates can be tuned by mutating the fourth ligand of type I copper. Further, mutation at the Cys ligand gives a vacant type I copper center and traps the reaction intermediate during the four-electron reduction of dioxygen.

show abstract

“…47 This process is calculated to have a barrier of ~ 8.5 kcal/mol which is reasonable given that the experimental barrier for the conversion of NI to resting is 8.8-13.9 kcal/mol. 48 Importantly, this rate of conversion (k = 0.05 s −1 ) 27 is orders of magnitude slower than the turnover rate of the enzyme (k= 350 s −1 ). 49 Therefore, NI is actually the catalytically relevant fully oxidized form of the MCOs.…”

Section: Ni Conversion To Resting: Interconvesion Of the Two Fully Oxmentioning

confidence: 99%

O2 Reduction to H2O by the multicopper oxidases

2008

View full text Add to dashboard Cite

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...

show abstract

Spectroscopic and Kinetic Studies on the Oxygen-centered Radical Formed during the Four-electron Reduction Process of Dioxygen byRhus vernicifera Laccase

Cited by 64 publications

References 33 publications

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

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

Structure and function of type I copper in multicopper oxidases

O2 Reduction to H2O by the multicopper oxidases

Contact Info

Product

Resources

About