Ho

Figure 1. Proposed mechanisms of oxidation of o-diphenol (cycle A, for catechol) and monophenol (cycle B) for Neurospora crassa PPO. Source: Adapted from Refs. 2 and 44.

on the B cycle). Subsequently, the o-position of the monophenol is hydroxylated by an oxygen atom of the 02 of the 02-PPO-monophenol complex to give catechol, which then dissociates to give deoxy PPO, to complete the cycle. Only the first cycle of hydroxylation on a monophenol requires starting at the met PPO; all subsequent cycles begin with deoxy PPO (2).

Because copper is the prosthetic group, the catalytic activity of PPOs is based on the cupric-cuprous valency change. On isolation of the enzyme in its natural state, the copper is in the cuprous form, but in the presence of o-dihydroxy phenols, the copper would be oxidized to cupric form. The substrate is oxidized by losing two electrons and two protons. The two electrons are taken up by the copper of the enzyme, which then passes into the cuprous state. The cuprous enzyme rapidly transfers the two electrons to oxygen, which immediately forms water with the two protons that were liberated, and the enzyme returns to the cupric state. This two-state reaction can be represented by

Recent understanding of the complete amino acid sequence of fungal PPO and the site of interaction with the phenolic substrate made it possible to create models that include a binuclear center. Histidine residues appear to lignand at least one of the two active sites of the copper atoms. Monophenols bind to one of the Cu2+ atoms, whereas diphenols bind to both of them, as shown in Figure 1 (59,60). Catechol oxidase can undergo conformational changes induced by the substrate 02 and by pH. The changes in conformation are accompanied by changes in the Km of the enzyme for both its substrates, oxygen and diphenol.

A two-step reaction mechanism by laccase has been reported (61). The first step in the oxidation of quinol by lac-

case was the formation of the semiquinone, with transfer of an electron from substrate to the copper in the enzyme. The second step was a nonenzymatic disproportionation reaction between two semiquinone molecules to give one molecule of quinone and one of quinol. The function of copper and electron transfer in the reaction mechanism has been studied by using inorganic ions, electron paramagnetic resonance, and spectrophotometric methods (3,62).

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