Vol. 36, No. 6, 1969 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNlCAllONS DIRECT DEMONSTRATION OF SUPEROEIDE ANION PRODUCTION DURING THE OXIDATION OF REDUCED FLAVIN AND OF ITS CATALYTIC DECOMPOSITION BY ERYTRROCUPREIN1 David Ballou, Graham Palmer, and Vincent Massey Department of Biological Chemistry, and Biophysics Research Division Institute of Science and Technology, University of Michigan, Ann Arbor, Michigan Received July 11, 1969 SUMMARY The oxidation of reduced flavins by molecular oxygen at neutral to alkaline pH produces substantial yields of the superoxide anion, 0 '. This species is rapidly destroyed by catalytic quantities of the coppgr protein, erythrocuprein, and by stoichiometric quantities of ferricytochrome 2. The involvement of'superoxide anion in the aerobic reduction of cytochrcme 2 catalyzed by xanthine oxidase and other metalloflavoproteins has been strongly suggested by the work of Fridovich and his colleagues (1,2,3). More recently, Knowles et al. -- (4) have demonstrated directly, by means of the rapid-freezing EPR (electron paramagnetic resonance) technique (5), that 02L is indeed a product of the oxidation of substrate-reduced xanthine oxidase. McCord and Fridovich (6) have recently provided strong evidence that erythrocuprein is a potent superoxide dismutase and that it inhibits the xanthine oxidase catalyzed aerobic reduction of cytochrome c. In both the work of Knowles et al. (4) and the earlier work of Fridovich et al -- --- it was suggested that the iron-sulfur component, rather than the flavin moiety, of these enzymes is responsible for the observed one electron reduction of oxygen. However, the observation that many metal free flavoproteins can catalyze an oxygen-dependent substrate-linked reduction of cytochrome 2 (7), which is inhibited by erythrocuprein, has led to the suggestion that reduced flavin can reduce oxygen to superoxide anion. 1 Supported by NIH Grants GM 12176 and GM 11106, NIH Predoctoral Fellowship GM 39-480 (D.B.) and Career Development Award GM 31-213 (G.P.) 898