Background: Chloroperoxidase (CPO) is the most versatile of the hemethiolate proteins, catalyzing the chlorination of activated CH bonds and reactions reminiscent of peroxidase, catalase, and cytochrome P450. Despite 30 years of continuous efforts, no intermediates of the enzyme's catalytic cycle have been identified except for compound I. Thus, in the absence of conclusive evidence it is generally believed that the halogenation of substrates proceeds by means of ‘free HOCl’ in solution.
Results: The pH profile of chloroperoxidase from Caldariomyces fumago revealed a new active-site complex that can be detected only at pH 4.4. According to ultra-violet (UV) spectroscopy, and by comparison with suitable enzyme models, this intermediate is the HOCl adduct of the iron(III) protoporphyrin(IX). Inactivation of chloroperoxidase by diethyl pyrocarbonate, which interrupts the proton shuttle by modification of the distal histidine, led to the formation of the −OCl adduct of the iron complex, which was identified by comparison with a corresponding active site analogue.
Conclusions: The availability of enzyme models of heme-thiolate proteins allowed the identification by UV spectroscopy of both the −OCl adduct and the HOCI adduct of the iron(III) protoporphyrin(IX) of chloroperoxidase. The existence of these previously elusive intermediates suggests that the chlorination catalyzed by CPO, and its corresponding active site analogue, proceeds by Cl+ transfer from the HOCl adduct to the substrate bound in the distal pocket of the enzyme.
