Active site models for galactose oxidase and related enzymes
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+)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].
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(9−)]+ 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
References (47)
- et al.
Trends in Biochemical Science
(1998) - et al.
FEBS Lett.
(1993) - et al.
J. Biol. Chem.
(1962) - et al.
J. Mol. Biol.
(1994) - et al.
J. Biol. Chem.
(1988) - et al.
J. Biol. Chem.
(1989) - et al.
J. Biol. Chem.
(1990) - et al.
Biophys. J.
(1993) - et al.
J. Biol. Chem.
(1996) - et al.
J. Mol. Biol.
(1994)
Biochim. Biophys. Acta
Chem. Rev.
Nature
Biochemistry
J. Am. Chem. Soc.
Biochemistry
J. Am. Chem. Soc.
J. Am. Chem. Soc.
Biochemistry
Proc. Natl. Acad. Sci. USA
J. Phys. Chem.
Cited by (144)
Tuning the locus of oxidation in Cu-diamido-diphenoxo complexes: From Cu(III) to Cu(II)-phenoxyl radical
2018, Inorganica Chimica ActaCitation Excerpt :In the last fifteen years [3], chemists have succeeded in generating and characterising persistent Cu(II)-phenoxyl radical complexes, providing a better understanding of the electronic, chemical, and even structural [4,5a] properties of the active form of GO. Recently, in [CuII-salen]+ species, a tuning of the locus of the radical has been achieved through ligand backbone substitutions, yielding Class III (fully delocalised π-radical) or Class II (localised phenoxyl radical) Cu(II)- radical complexes [3e,5]. Remarkably, in the case of Cu(1,2-salen), the one-electron oxidation results in a high-valent, diamagnetic Cu(III) species in the solid state; whilst in solution a temperature-dependent valence tautomerism exists between the Cu(III) and the Cu(II)-radical oxidised forms [5b].
Photochemical direct perfluoroalkylation of phenols
2015, TetrahedronDiversity of oxidation state in copper complexes with phenolate ligands
2024, Dalton TransactionsAltering the Localization of an Unpaired Spin in a Formal Ni(V) Species
2024, Chemistry - A European Journal
- 1
Also corresponding author.