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.
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