Stabilization of Lactate Oxidase Flavin Anion Radical

The red-colored flavin anion radical of lactate oxidase was formed by photochemical reduction. The radical was found to be reoxidized readily by molecular oxygen, with a rate constant of 4 X lo5 M "I min" at pH 7.0, 25°C. On mixing the radical under anaerobic conditions with pyruvate, a change in spectrum typical of charge transfer interaction was found. The complex was composed of 1 eq of pyruvate per eq of enzyme flavin radical, with a Kd of 1.4 x lo-' M. The complex was sufficiently stable at 0°C that it could be isolated free of excess keto acid by gel filtration under aerobic conditions. The isolated complex appears to be quite inert to oxidation by 02; the slow reoxidation which was observed was due to the slow dissociation of the complex and the subsequent fast reaction of O2 with the radical not in complex. The observed rate of oxidation is markedly dependent on temperature, with an activation energy of 25 kcal per mol.