Glyoxal Oxidase from Phanerochaete chrysosporium Is a New Radical-Copper Oxidase

Mei M Whittaker,Philip J Kersten,Nobuhumi Nakamura,Joann Sanders-Loehr,Elizabeth S Schweizer,James W Whittaker

doi:10.1074/jbc.271.2.681

Abstract

A free radical-coupled copper complex has been identified as the catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of spectroscopic and biochemical studies. The native (inactive) enzyme is activated by oxidants leading to the elimination of the cupric EPR signal consistent with formation of an antiferromagnetically coupled radical-copper complex. Oxidation also leads to the appearance of a substoichiometric free radical EPR signal with an average g value (gav = 2.0055) characteristic of phenoxyl tau-radicals arising from a minority apoenzyme fraction. Optical absorption, CD, and spectroelectrochemical measurements on the active enzyme reveal complex spectra extending into the near IR and define the redox potential for radical formation (E 1/2 = 0.64 V versus NHE, pH 7.0). Resonance Raman spectra have identified the signature of a modified (cysteinyl-tyrosine) phenoxyl in the vibrational spectra of the active complex. This radical-copper motif has previously been found only in galactose oxidase, with which glyoxal oxidase shares many properties despite lacking obvious sequence identity, and catalyzing a distinct reaction. The enzymes thus represent members of a growing class of free radical metalloenzymes based on the radical-copper catalytic motif and appear to represent functional variants that have evolved to distinct catalytic roles.

Highlights

A free radical-coupled copper complex has been identified as the catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of spectroscopic and biochemical studies
The oxidizing peroxide cosubstrate for this reaction must be generated in situ for efficient turnover of extracellular lignin peroxidase, a function performed by glyoxal oxidase, which catalyzes the oxidation of a number of aldehyde
Spectroscopic and biochemical characterization of the active sites of glyoxal oxidase and galactose oxidase reveals an amazing degree of similarity between two proteins essentially lacking any obvious sequence homology and catalyzing distinct reactions

Summary

Introduction

A free radical-coupled copper complex has been identified as the catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of spectroscopic and biochemical studies. Resonance Raman spectra have identified the signature of a modified (cysteinyl-tyrosine) phenoxyl in the vibrational spectra of the active complex This radical-copper motif has previously been found only in galactose oxidase, with which glyoxal oxidase shares many properties despite lacking obvious sequence identity, and catalyzing a distinct reaction. The active site structure of galactose oxidase as revealed by crystallography [17] is illustrated in Scheme 1, showing the coordination of the copper ion by 2 histidine residues (His-496 and His-581), a simple tyrosinate (Tyr-495), and a covalently modified tyrosine (Tyr-272), cross-linked to a cysteinyl residue (Cys-228) to form a new, dimeric amino acid (cysteinyl-tyrosine) This feature has been identified by spectroscopic and modelling studies as the radical-forming site in galactose oxidase (18 –20). Spectroscopic and biochemical comparison of the two proteins suggests that glyoxal oxidase and galactose oxidase are functional variants catalyzing distinct reactions at nearly identical active sites

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Conclusion