Human Heme Oxygenase Oxidation of 5- and 15-Phenylhemes

Abstract

Human heme oxygenase-1 (hHO-1) catalyzes the O 2 -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 ␣-meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IX␣. Surprisingly, a 15-methyl substituent caused exclusive cleavage at the ␥-meso-rather than at the normal, unsubstituted ␣-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 IX␣ and oxidizes 15-phenylheme at the ␣-meso position to give 10-phenylbiliverdin IX␣. The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O 2 and the other from water. The 2.29-and 2.11-Å 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 Lys 18 and Lys 22 . In the 5-phenylhemehHO-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 Met 34 , Phe 214 , and residues 26 -42 near the ␣-meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.Heme 1 oxygenase, the rate-limiting enzyme in the heme degradation pathway, oxidizes heme to biliverdin, carbon monoxide, and free iron (1) (Fig. 1). In humans and other mammals, the heme is cleaved exclusively at the ␣-meso position to give biliverdin IX␣. The biliverdin then is reduced to bilirubin by biliverdin reductase before it is conjugated with glucuronic acid and excreted in the bile (2). Bilirubin is a powerful physiological antioxidant (3), but is neurotoxic at the high concentrations found in Criggler Najar patients and neonatal jaundice (4). CO, the second product of the reaction, is a gaseous messenger molecule that is thought to be involved in apoptosis (5), atherosclerosis (6), psoriasis (7), vascular constriction (8), cellular protection (9), and chronic renal inflammation (10). Finally, the ferrous iron released by heme oxygenase is normally recycled and serves as the major source of this metal for hemoglobin and other hemoprotein synthesis, as only 1-3% of the iron utilized daily in the synthesis of red blood cells is obtained from the diet (11). Thus, in addition to its other functions, heme oxygenase plays a vital role in maintaining iron homeostasis.In mammals, there are three heme oxygenase isoforms, namely HO-1, HO-2, and HO-3. HO-1 is inducible and present in highest concentration in spleen and liver. It is a stress pro...

Isoporphyrin Intermediate in Heme Oxygenase Catalysis

“…The results showed that one atom of labeled oxygen was incorporated from water and one from the peroxide or dioxygen into the corresponding para-substituted benzoic acid product, as previously reported for the oxidation of ␣-meso-phenylmesoheme (supplemental Fig. S4) (23). No oxygen-18 was incorporated into biliverdin from H 2 18 O 2 , in accord with the fact that the two lactam oxygens arise from two different molecules of O 2 (supplemental Fig.…”

“…CO Formation Assay-Horse myoglobin (Sigma) was used as a CO trap according to the protocol previously described (23). The reactions (1 ml) consisted of 10 M arylmesoheme-hHO-1 complex, 60 nM CPR, and 100 M NADPH in standard buffer and were allowed to proceed for 20 min in a sealed container before the addition of myoglobin via syringe to a final concentration of 4 M.…”

“…The dimethyl esterified biliverdin isomers were prepared from the corresponding heme by coupled oxidation in the presence of pyridine, O 2 , and ascorbic acid according to published procedures (27,28 (29). Enzymes-Truncated hHO-1 lacking the 23 C-terminal residues was expressed and purified according to published procedures (23,30,31). This truncated protein is referred to as hHO-1 throughout the paper.…”

“…Previous studies have investigated the oxidation of hemes in which the ␣-meso carbon bears a methyl, formyl, or phenyl substituent (21)(22)(23). Surprisingly, phenyl-substituted hemes readily bind to the hHO-1 active site due to the presence of a hydrophobic cavity, formed by residues Met-34, Phe-37, and Phe-214, that is postulated to function as a CO trapping site (24).…”

“…More recently, benzoic acid has been identified as a product of the hHO-1-catalyzed oxidation of ␣-meso-phenylheme. An additional intermediate is required to account for benzoic acid formation (23).…”