Horseradish peroxidase-catalyzed two-electron oxidations. Oxidation of iodide, thioanisoles, and phenols at distinct sites.

The atypical two-electron oxidation of thioanisole and its p-methyl, p-methoxy, and p-nitro analogues by horseradish peroxidase, contrary to earlier reports, stereoselectively produces the (S) sulfoxides in 60-70% enantiomeric excess. Horseradish peroxidase reconstituted with delta-meso-ethylheme has little peroxidase (guaiacol oxidizing) activity, as previously reported, but exhibits increased sulfoxidation activity. Difference spectroscopy shows that guaiacol binds to delta-meso-ethylheme-reconstituted horseradish peroxidase even though it is essentially not oxidized. In contrast, horseradish peroxidase reconstituted with delta-meso-methylheme is active in both reactions. Studies with H(2)18O2 show that the oxygen in the sulfoxide produced by delta-meso-ethylheme-reconstituted horseradish peroxidase derives, as it does in the reaction catalyzed by the native enzyme, primarily from the peroxide. Preincubation of horseradish peroxidase with phenylhydrazine, which modifies the protein, suppresses peroxidase activity but does not inhibit thioanisole sulfoxidation. On the other hand, the oxidation of iodide is blocked by reconstitution of horseradish peroxidase with delta-meso-ethylheme or preincubation with phenylhydrazine. Noncompetitive kinetics are observed for the inhibition of guaiacol and iodide oxidation by thioanisole and of guaiacol oxidation by iodide. The kinetic data and the differential inhibitory effects of delta-meso-ethylheme reconstitution and phenylhydrazine preincubation indicate that thioanisole and iodide, both of which undergo net two-electron oxidations, are oxidized at sites distinct from each other and from that involved in the oxidation of guaiacol. Spectroscopic substrate binding studies provide support for distinct thioanisole, guaiacol, and iodide-binding sites. An active site model is proposed to rationalize the results.