Abstract
The copper enzyme galactose oxidase has been prepared in three distinct redox modifications; two of these represent nearly homogeneous preparations of active and inactive species which have been described previously, while the third has never before been reported. Preparation of these redox modifications as homogeneous species has permitted detailed spectroscopic and catalytic studies of each for the first time. We find that the form which has been extensively probed by EPR spectroscopy is devoid of catalytic activity and does not interact with substrate. The detailed characterization of oxidatively activated galactose oxidase and its anion interactions has led to a spectroscopic assignment of the copper oxidation state in this complex which indicates that the one-electron redox process which converts the inactive form to catalytically active enzyme is associated with oxidation of the protein rather than the metal center as has been proposed previously. This oxidation step is required for catalytic activity and is the basis of the two-electron redox reactivity for the enzyme active site: anaerobic addition of hydroxylic substrates results in reduction of the two-electron redox unit, and the spectral features associated with both the copper ion and the non-metal redox center are eliminated, apparently forming a cuprous site. The two-electron reactivity resulting from protein participation in redox catalysis has important implications in this and other mechanisms where oxygen reduction occurs at a mononuclear metal ion active site.
Highlights
The copper enzyme galactose oxidase has been pre- the absence of 02.Recent studies show that, in addition to pared in three distinctredox modifications; two of catalyzing the oxidation of alcohols, the enzyme further conthese represent nearly homogeneous preparations of verts aldehydes to thecorresponding carboxylates [8].In spite active and inactivespecies which have been described of the attention that thiesnzyme has received, no significant previously, while the third hasnever before been reported
We find that the form which has been extensively probed by EPR spectroscopy is devoid of catalytic activity and does not interact with substrate
The detailed characterization of oxidatively activatedgalactose oxidase and its anion interactions has led to a spectroscopic assignment of the copper oxidation state results have appeared from spectroscopic approaches to provide insight into theredox mechanism, mainly because of an unpredictable variability in the spectral properties of the native enzyme
Summary
Vol 263, No 13, Issue of May 5, pp. 6074-6080, 1988 Printed in U S A. Ase [13] and hemocyanin [14].The structural basis for the unusual reactivity of galactose oxidase has never been identified, involvement of higher oxidation states of copper has been proposed [11].Our interests in the twoelectron redox mechanism and the structural origins of this unusual reactivity haveled us to investigate in detail the redox modifications of the active site which can be used to prepare homogeneous forms for spectral analysis. This spin standard avoids problems with Cuz+dimerization which would result in signal intensity artifactsand exhibits amore homogeneousEPR signal than other complexes more commonly used as spin standards.' Accurate quantitation required nonsaturating power levels, uniform, stable, and well defined sample temperature, uniform sample position within the microwave cavity, and application of the Aasa correction [17] for field-swept signal intensities
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
