Effect of monovalent anions on the mechanism of phenol hydroxylase.

The mechanism of phenol hydroxylase (EC 1.14.13.7) has been studied by steady state and rapid reaction kinetic techniques. Both techniques give results consistent with the Bi Uni Uni Bi ping-pong mechanism proposed for other flavin-containing aromatic hydroxylases. The enzyme binds phenolic substrate and NADPH in that order, followed by reduction of the flavin and release of NADP+. A transient charge transfer complex between reduced enzyme and NADP+ can be detected. Molecular oxygen then reacts with the reduced enzyme-substrate complex. Two to three flavin-oxygen intermediates can be detected in the oxidative half-reaction depending on the substrate, provided monovalent anions are present. Oxygen transfer is complete with the formation of the second intermediate. Based on its UV absorption spectrum and on the fact that oxygen transfer has taken place, the last of these intermediates is presumably the flavin C(4a)-hydroxide. Monovalent anions are uncompetitive inhibitors of phenol hydroxylase. The mechanistic step most affected is the dehydration of the flavin C(4a)-hydroxide to give oxidized enzyme. Chloride also kinetically stabilizes the blue flavin semiquinone of phenol hydroxylase during photoreduction. These data suggest binding of monovalent anions results in stabilization of a proton on the N(5) position of the flavin.