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J. Biol. Chem., Vol. 280, Issue 4, 2797-2806, January 28, 2005

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Regiospecificity Determinants of Human Heme Oxygenase

DIFFERENTIAL NADPH- AND ASCORBATE-DEPENDENT HEME CLEAVAGE BY THE R183E MUTANT^*

Jinling Wang, Latesh Lad, Thomas L. Poulos, and Paul R. Ortiz de Montellano¶

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280 and the Department of Molecular Biology and Biochemistry, Department of Physiology and Biophysics, Department of Chemistry and Center for Chemical and Structural Biology, University of California, Irvine, California 92697-3900

The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IX. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IX (79 ± 4%), IX (19 ± 3%), and a trace of IX. In the presence of superoxide dismutase and catalase, the yield of biliverdin IX is decreased to 8 ± 1% with a corresponding increase in biliverdin IX. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II)·NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

Received for publication, September 30, 2004

The atomic coordinates and structure factors (codes 1XK2 and 1XK3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grants DK30297 (to P. R. O. M.) and GM33688 (to T. L. P.). 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.

^¶ To whom correspondence should be addressed: University of California, San Francisco, 600 16th St., N572D, San Francisco, CA 94143-2280. Tel.: 415-476-2903; Fax: 415-502-4728; E-mail: ortiz{at}cgl.ucsf.edu.

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