Active site models for galactose oxidase and related enzymes

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Abstract

Redox interaction between a transition-metal ion and a redox active amino acid side chain such as the phenol group of tyrosine in several enzymatic systems has been discovered to play a crucial role in biologically important processes. The tyrosyl radical, which directly coordinates to the copper ion center, has recently been found in the active sites of galactose oxidase (GAO) and glyoxal oxidase (GLO). In this article, model studies on the active site of the enzymes are reviewed by summarizing reported information about the physicochemical properties and the redox functions of the Cu(II) and Zn(II) complexes of the phenolate and phenoxyl radical forms of the cofactor models as well as the organic cofactor models themselves.

Introduction

The tyrosyl radical has now been well-recognized to play a crucial role in several enzymatic redox processes [1], [2], [3]. The R2 subunit of non-heme diiron enzyme ribonucleotide reductase from E. coli is one of the most well-characterized examples of such enzymes, where the tyrosyl radical, derived from Tyr 122 via the one-electron oxidation by the adjacent Fe(III)/Fe(IV)-oxo species (so-called intermediate X), initiates the nucleotide-reduction process [1], [2]. Tyrosyl radicals are also involved as important intermediates in the redox processes of prostaglandin H synthase and photosystem II, in which (porp+radical dot)Fe(IV)O (porp=protoporphyrin IX) and a high valent manganese cluster are responsible for the tyrosyl radical formation, respectively [1], [2]. The tyrosyl radical, which directly coordinates to the copper ion center, has recently been found in the active site of galactose oxidase (GAO, EC 1.1.3.9) that catalyzes the oxidation of d-galactose and primary alcohols to the corresponding aldehydes coupled to the reduction of O2 to H2O2 (Eq. (1)) [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15].RCH2OH+O2→RCHO+H2O2

The crystal structure of galactose oxidase at 1.7 Å resolution has clearly shown that the tyrosine residue (Tyr 272) is covalently bound to the sulfur atom of adjacent Cys 228 at the α-position of the phenol ring as illustrated in Scheme 1 [6], [7]. This built-in organic cofactor serves as a one-electron redox center by shuttling between the phenol and phenoxyl radical forms during the course of the redox cycle; the alcohol-oxidation and the O2-reduction [8], [9], [10], [11], [12]. Thus, the active species (fully oxidized state) of the enzyme is the Cu(II)–phenoxyl radical of Tyr 272 that can oxidize alcohols to the corresponding aldehydes by the following mechanism: (i) deprotonation from the OH group of the bound substrates by the phenolate group of Tyr 495, (ii) inner-sphere electron transfer from the deprotonated substrate to Cu(II), and (iii) α-hydrogen atom abstraction of the resulting ketyl radical by the phenoxyl radical of Tyr 272 species (the ordering of electron transfer and hydrogen atom abstraction steps could be reversed) [11], [12], [13], [14], [15]. It is further proposed that the fully oxidized state is reproduced from the fully reduced state [Cu(I)–phenol] by the reaction with molecular oxygen that is transformed into hydrogen peroxide as shown in Eq. (1). The interconversion between Cu(I) and Cu(II) states has also been demonstrated by X-ray absorption spectroscopy [16], [17]. Such a phenoxyl radical–copper catalytic motif has also been found in glyoxal oxidase (GLO) from Phanerochaete chrysosporium and in the prokaryotic FbfB protein [18], [19], [20].

The development of synthetic analogues of metalloenzyme active sites has provided valuable insight into structures, physicochemical properties, and functions of active intermediates in enzymatic reactions, which are often obscured by the huge peptide backbones of the native enzymes. Model studies on GAO and the related enzymes have also given valuable information about (i) the electronic effects of the thioether group of the cofactor, (ii) physicochemical properties of phenoxyl radical species of the cofactor both in the metal-free form and in the metal complexes, and (iii) the catalytic alcohol oxidation by Cu(II)–phenoxyl radical complexes. The purpose of this review article is to summarize such studies. Copper complexes of simple phenol derivatives containing a metal binding site are not included in this article.

Section snippets

Neutral and anionic (phenolate) forms

One of the most interesting features of the enzyme is the existence of the thioether linkage between Tyr 272 and Cys 228 (Scheme 1). Then a question arises why galactose oxidase and the related enzymes employ such a modified amino acid residue instead of a simple tyrosine. The redox potential of galactose oxidase is estimated to be 400–500 mV vs. NHE [10], that is significantly lower than that of free tyrosine in solution (930 mV) or tyrosine in enzymatic systems (760–1000 mV) [21], [22]. Such

Copper(II)–phenolate complexes (resting state model)

The first copper(II) complex of the cofactor model was reported by Whittaker and co-workers in 1993 [26], [31]. It is a ternary complex consisting of a copper(II) ion, 1 as the cofactor model, and N,N,N′,N′,N′′-pentamethyldiethylenetriamine (PMDT) as a supporting ligand, [CuII(PMDT)(1)](ClO4)·CH3OH [26]. The copper complex has a square pyramidal structure in which basal plane is occupied by three nitrogen atoms of PMDT and the phenolate oxygen of 1 [26]. The sulfur atom of the methylthio

Copper(II)–phenoxyl radical complexes (active form model)

Six types of Cu(II)–phenoxyl radical complexes have so far been reported as models of the active form of GAO (Table 3) [35], [38], [44], [45], [46]. Chemical or electrochemical one-electron oxidation of the corresponding Cu(II)–phenolate complexes has provided relatively stable Cu(II)–phenoxyl radical complexes. All the Cu(II)–phenoxyl radical complexes exhibit a characteristic absorption band around 400 nm, which appears in the somewhat lower wavelength region than that of the native enzymes

Alcohol-oxidation by phenoxyl radical complexes

[CuII(9radical dot)]+ developed as a functional model of GAO has shown to catalyze oxidation of primary alcohols to the corresponding aldehydes [37], [38]. Turnover numbers more than 103 has been achieved using O2 as an electron acceptor [38]. A significantly distorted square planar coordination geometry with the N2O2 donor set was proposed to be essential for enhancing the substrate binding process and the alcohol-oxidation reaction [38]. Namely, the substrate binding leads to a five coordinate square

Summary

In this mini review article, the recent model studies on the active sites of galactose oxidase (GAO) and glyoxal oxidase (GLO) have been summarized. Studies on the physicochemical properties of organic cofactor models in both the phenol (phenolate) and phenoxyl radical forms have indicated that the thioether group of the cofactor has the 2pπ–3dπ electron conjugative effect as well as the electron-donating nature, stabilizing the negative charge on the phenolate oxygen. The electron-sharing

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