Characterization of the Electron Acceptors of Old Yellow Enzyme: Mechanistic Approach to the Mode of One Electron Transfer from the Enzyme to Menadione or Dyestuffs

Molecular oxygen or cytochrome c has been described as the electron acceptor of the reaction of old yellow enzyme with NADPH. In this study, menadione was found to be a sensitive electron acceptor of the reaction under aerobic as well as anaerobic conditions. The Km value of menadione for old yellow enzyme is as low as 2−3 × 10−7 M in the presence or absence of superoxide dismutase. The rate enhancement of the cytochrome c reduction of old yellow enzyme with NADPH was about eight times in the presence of menadione. The rate increment was slightly higher under aerobic than anaerobic conditions. The rate enhancement by menadione enabled sensitive determination of the enzyme activity in the assay system, which contained NADPH, cytochrome c, menadione, and old yellow enzyme. In the reaction course, the semiquinone species of menadione was trapped by the reaction with t-butyl-α-phenylnitrone. The radical adduct was detected on EPR. The dyestuff, 2, 6-dichlorophenolindophenol, was found to be reduced ineffectively even in the presence of menadione; moreover, it was inhibitory in the NADPH consumption reaction. Methylene blue or Lauth's violet, known to be capable of semiquinone formation, also behaved, like menadione, as a mediator of electron transport to cytochrome c. On the basis of the experimental results, the occurrence of the one electron transfer of the old yellow enzyme reaction was emphasized.