Involvement of the binuclear copper site in the proteolytic activity of polyphenol oxidase

Plant polyphenol oxidase (PPO) is apt to degrade during and even after purification. We developed a method to stabilize PPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethylene glycol at pH 6.5. The protein slowly degraded by itself when the stabilizing reagents were removed. Ascorbate and/or H2O2 accelerated the degradation. The ascorbate-induced degradation was inhibited by catalase, suggesting that H2O2 is generated through reduction of PPO by ascorbate. It is likely that dissolved oxygen is converted to peroxide through two-electron reduction by the reaction center of PPO, binuclear Cu site, and a Fenton-type reaction occurred on it. This understanding was supported by the finding that the H2O2-induced degradation was inhibited by metal-chelators as well as by polyphenolic substrate of PPO. Considering the postulated mechanism of the self-degradation of PPO, we re-examined the degradation of the 23-kDa protein of PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38: 179]. The obtained results suggested that the 23-kDa protein triggers the active oxygen production by the binuclear Cu site, probably as reductant, and receives the radical species preferentially to the polypeptide moiety of PPO.