The percentage of one-electron reduction of p-benzoquinone and oxygen was measured in various reactions of xanthine-oxidizing enzymes using cytochrome c as a scavenger. The mechanism of p-benzoquinone reduction was different in milk xanthine oxidase (pancreatintreated) and chicken liver xanthine dehydrogenase when xanthine was used as an electron donor. Similar differences in the reduction mechanism were observed in the type O and type D forms of the reversibly convertible enzyme prepared by the method of Battelli et al. ((1973) Biochem. J. 131, 191-198). The mechanism changed when xanthine was replaced by NADH. When NADH. was used as the electron donor, no significant difference was observed in the mechanism of p-benzoquinone reduction among the four enzymes.
Sulfhydryl groups were found to play a crucial role in the xanthine-NAD+ reductase activity of the type D enzyme and xanthine dehydrogenase. The reductase activity disappeared upon treatment with pCMB. The reductase activity was sensitive to DTNB in the type D enzyme but not in the xanthine dehydrogenase. The oxidase activity of the type D enzyme was restored by autoxidation of the sulfhydryl groups but not by reaction of the enzyme with DTNB or pCMB. It was concluded that the formation of S-S bond (s) was essential for the oxidase activity and also for the oxidase-type mechanism of p-benzoquinone reduction in the xanthine-p-benzoquinone reactions.