Studies of Aldehyde Oxidase by Electron Paramagnetic Resonance Spectroscopy

The kinetics of appearance and disappearance of the electron paramagnetic resonance (EPR) signals of aldehyde oxidase under various conditions were studied by a rapid freezing technique. Electron spin relaxation of the paramagnetic flavin and molybdenum species was monitored during these experiments. The kinetics of reduction and reoxidation of molybdenum, iron, and flavin, revealed by EPR measurements, were compatible with the participation of these substances in the normal catalytic cycle of this enzyme. In the steady state molybdenum is partly reduced, whereas the iron and flavin components are largely oxidized. Hence, reduction of molybdenum and oxidation of flavin and iron are relatively rapid reactions. No unambiguous evidence for a linear sequence of electron carriers was obtained, but a sequence of electron transfer from substrate to molybdenum, flavin, and iron is compatible with most results. The results of the electron spin relaxation measurements indicate a multiplicity of subtile interactions among the electron carriers involved, dependent on time of reaction, type of substrate, and degree of reduction. The multiple complexities in the reaction patterns of enzymes incorporating several electron carriers, as they become apparent in studies of the kind presented here, are discussed.