Horseradish Peroxidase Catalyzed Two Electron Oxidations

THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol ,268. No. 3, Issue of January 25, pp. 1637-1645,1993 Printed in U.S.A .

Horseradish Peroxidase-catalyzed Two-electronOxidations
OXIDATIONOFIODIDE,THIOANISOLES,ANDPHENOLSATDISTINCT

SITES*
(Received for publication, July 2, 1992)

Robert Z. Harris, SherriL. Newmyer,and Paul Ortiz de R. Montellano$
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, Sun Francisco, California 94143-0446

The atypical two-electron oxidation of thioanisole from two substrate molecules reduces the porphyrin radical and its p-methyl, p-methoxy, and p-nitro analogues cation first to the by ferryl porphyrin (PFe” = 0)species known horseradishperoxidase, contrary to earlier reports, as compound 11, and subsequently to the ferric resting state. stereoselectively produces the (8) sulfoxides in 60- Normally, the rateof the first electron abstraction is approx70% enantiomeric excess. Horseradish peroxidase re- imately 10 times faster than that of the second, so that the constituted with 6-meso-ethylheme has little peroxichromophore of compoundI1 is usually observed during as previously re- steady-state catalytic turnover (4, 5 ) . Despite the structural dase (guaiacol oxidizing) activity, ported, but exhibits increased sulfoxidation activity. proposed similarity of the ferryl complexof compound I to the Difference spectroscopy shows that guaiacol binds to catalytic species of cytochrome P-450 and towell-character6-meso-ethylheme-reconstituted horseradish peroxidase even though itis essentially not oxidized.In con- ized ferryl metalloporphyrin complexes (6, 7), horseradish trast, horseradishperoxidasereconstitutedwith 6- peroxidase does not generally transfer theferryl oxygen to its meso-methylheme is active in both reactions. Studies substrates. It does not, for example, catalyze the epoxidation of styrene (8) orbutadiene(9),activated olefins that are with H,“02 show that the oxygen in the sulfoxide readily oxidized by cytochrome P-450 monooxygenases and produced 6-meso-ethylheme-reconstituted by horsemetalloporphyrin monooxygenase models (4, 5).’ To explain radish peroxidase derives, as it does in the reaction this divergence incatalyticfunction of nominallyrelated catalyzed by the native enzyme, primarily from theperoxide.Preincubation of horseradishperoxidase catalytic species, we have proposed that substrates interact withphenylhydrazine,which modifies theprotein, with the b-meso edge of the heme group of horseradish persuppressesperoxidaseactivitybutdoesnotinhibit oxidase rather than with the ferryl oxygen itself (10). This thioanisole sulfoxidation. On the other hand, the oxi- proposal derives much ofits support from the observation dation of iodide is blocked by reconstitution of horse- that catalytic oxidation of several substrates by horseradish radish peroxidase with 6-meso-ethylheme or preincu- peroxidase results in alkylation of the 6-meso carbon rather bation with phenylhydrazine. Noncompetitive kineticsthan, as in cytochrome P-450, theiron or theporphyrin a r e observed for theinhibitionof guaiacol and iodide group (10-13). Furthermore, oxidation by thioanisole and of guaiacol oxidation by nitrogens of the prosthetic heme horseradish peroxidase reconstitutedwith a 6-meso-ethyliodide. The kinetic data and the differential inhibitory to peroxidase effects of 6-meso-ethylheme reconstitution and phen- heme group appears react with H202 but has no activity (13). An active site inwhich peroxidase substrates ylhydrazinepreincubationindicate that thioanisole bind near the 6-meso edge but are prevented by a protein and iodide, both of which undergo net two-electron barrier from interactingwiththe ferryl oxygen has been oxidations, are oxidized at sites distinct from each other and from thatinvolved in the oxidation guaia- postulated to rationalize the 6-meso edge sensitivity...