Abstract
Human heme oxygenase-1 (hHO-1) catalyzes the O2-dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the alpha-meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IXalpha. Surprisingly, a 15-methyl substituent caused exclusive cleavage at the gamma-meso-rather than at the normal, unsubstituted alpha-meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IXalpha and oxidizes 15-phenylheme at the alpha-meso position to give 10-phenylbiliverdin IXalpha. The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-A crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141-150 and in the proximal Lys18 and Lys22. In the 5-phenylheme-hHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26-42 near the alpha-meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.
Highlights
Heme1 oxygenase, the rate-limiting enzyme in the heme degradation pathway, oxidizes heme to biliverdin, carbon monoxide, and free iron [1] (Fig. 1)
To further characterize the mechanism governing the degradation of the meso-substituted heme, we have examined the Human heme oxygenase-1 (hHO-1)-catalyzed oxidation of the 5- and 15-phenylhemes (Fig. 2)
UV-visible Spectra of Heme-hHO-1, 5-hHO-1, and 15-hHO-1— Apo-hHO-1 was combined with heme, 5-phenylheme, or 15phenylheme and the resulting reconstituted heme oxygenase was immediately purified
Summary
Materials—5-Phenylheme, 15-phenylheme, and the biliverdin isomers derived from the dimethyl esters of 5-phenylheme and 15-phenylheme (Fig. 2) were prepared as reported previously (24 –26). HPLC Analysis of Biliverdin Isomers—Heme-hHO-1, 5-hHO-1, or 15-hHO-1 (45 nmol) was incubated with NADPH (3.4 mol) and P450 reductase (4.5 nmol) in standard buffer for 2 h at room temperature. HPLC Analysis of the Fragment Containing the ␣-Carbon and Phenyl Group—The heme-hHO-1, 5-hHO-1, or 15-hHO-1 complex (ϳ7 nmol) was incubated with NADPH (400 nmol) and P450 reductase (0.6 nmol) in standard buffer at room temperature for at least 1 h. HPLC was carried out at room temperature with a Varian 1090 solvent delivery system and a Hewlett-Packard 1040A detector, using the following conditions (solution A, H2O/0.05% trifluoroacetic acid; solution B, methanol/0.1% trifluoroacetic acid, flow rate, 1.0 ml/min): 10% B for 5 min, 10 –30% B in 0.1 min, 30 –55% B in 25 min, 55–95% B in 5 min, 95% B for 5 min, 95–10% B in 0.1 min, and 10% B for 15 min.
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