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J. Biol. Chem., Vol. 283, Issue 28, 19530-19539, July 11, 2008

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Isoporphyrin Intermediate in Heme Oxygenase Catalysis

OXIDATION OF -MESO-PHENYLHEME^*

John P. Evans, Fernando Niemevz, Graciela Buldain, and Paul Ortiz de Montellano¹

From the Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94158 and Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 956 (C1113AAD), Buenos Aires, Argentina

Human heme oxygenase-1 (hHO-1) catalyzes the O₂- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the -meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin -cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of -meso-phenylheme-IX, -meso-(p-methylphenyl)-mesoheme-III, and -meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593–42604), only the -biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H₂O₂ and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H₂O₂ identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H₂¹⁸O₂, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H₂O₂-generated hHO-1-isoporphyrin complex produced -biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation.

Received for publication, November 27, 2007 , and in revised form, May 16, 2008.

This paper is dedicated to Professor Elias J. Corey, a pioneer in the application of synthetic organic chemistry to the elucidation of biological problems, on the occasion of his 80th birthday.

^* This work was supported, in whole or in part, by National Institutes of Health Grant DK30297 (to PROM). This work was also supported by Universidad de Buenos Aires Grant B005 (to G. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ To whom correspondence should be addressed: Dept. of Pharmaceutical Chemistry, 600 16th St., San Francisco, CA 94158-2517. Tel.: 415-476-2903; E-mail: ortiz{at}cgl.ucsf.edu.

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