Participation of Carboxylate Amino Acid Side Chain in Regiospecific Oxidation of Heme by Heme Oxygenase

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)⅐NO 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 NADPHdependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPHcytochrome P450 reductase. Heme1 oxygenase is the key enzyme in the mammalian heme degradation pathway. It catalyzes the conversion of heme to biliverdin IX␣, CO, and free iron in a reaction that utilizes three molecules of molecular oxygen and seven electrons provided by NADPH-P450 reductase (see Fig. 1) (1, 2). The biliverdin is subsequently reduced to bilirubin by biliverdin reductase and is then excreted as the glucuronic acid conjugate (3). Heme oxygenase and its products are now known to contribute to diverse physiological protective mechanisms. First, biliverdin and bilirubin are potent antioxidants (4). Second, heme oxygenase removes nonspecifically bound heme, which is a source of noxious free radicals and other oxidizing species. Third, CO has anti-inflammatory effects mediated via the mitogen-activated protein kinase pathway (5, 6). Finally, heme oxygenase is essential for cellular iron homeostasis, because heme catabolism is by far the principal source of iron in the body (7). Recent studies have reported the potential importance of heme oxygenase and its products in the treatment of various disorders, including cardiovascular diseases, cancer, and allograft tissue rejection (8 -12). However, as found in CrigglerNajar patients and in neonatal jaundice (13), high concentrations of unconjugated bilirubin are neurotoxic.Three heme oxygenase isoforms, HO-1, HO-2, and HO-3, have been reported in humans and other mammals. HO-1, a stress-respon...

Section: Uv-visible Spectra Of the Heme⅐r183e Hho-1 Complex-thecontrasting

confidence: 43%

Section: Hemementioning

confidence: 99%

Regiospecificity Determinants of Human Heme Oxygenase

Wang

Lad

Poulos

et al. 2005

“…That the heme-propionate interactions remain unchanged, while the hydrophobic wall near the ␣-meso edge disorders indicates that the heme-propionate interactions must be very important in properly orienting the heme. The importance of propionate-protein interactions is supported by mutagenesis studies (56). Furthermore, the recently determined crystal structure of a bacterial heme oxygenase from P. aeruginosa, a HO enzyme that primarily hydroxylates the ␦-mesocarbon, shows that the novel regioselectivity is due to rotation of the heme by ϳ100° (57).…”

Section: Discussionmentioning

confidence: 70%

Human Heme Oxygenase Oxidation of 5- and 15-Phenylhemes

Wang

Niemevz

Lad

et al. 2004

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...

“…Mutation of Ser-142 shift the pK a value of the distal water ligand toward more basic values. 2 Lys-179 and Arg-183 appear to interact with the propionate carboxyl groups of the heme and may be important for proper orientation of the heme (47,48). We have therefore examined the binding of NO to the E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A mutants, all of which were heterologously expressed in Escherichia coli, purified, and found to have appropriate Soret maxima (Table II).…”

Section: No Binding To Mutant Ferric Hho-1-heme Complexes-as Shown Inmentioning

confidence: 99%

Interaction of Nitric Oxide with Human Heme Oxygenase-1

Wang¹,

Shen²,

Moënne‐Loccoz³

et al. 2003

NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe 3؉ -NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide⅐sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The (N-O) vibration of this complex detected by Fourier transform IR is only 4 cm ؊1 lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe 3؉ -NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k on for formation of the hHO-1-heme Fe 3؉ -NO complex is ϳ50-times faster, and k off 10 times slower, than for met-Mb, resulting in K d ‫؍‬ 1.4 M for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K d value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO. Nitric oxide (NO)1 functions as a signaling molecule in a diversity of physiological responses, including vasodilation and regulation of normal vascular tone, neuronal signal transmission, cytotoxicity against pathogens and tumors, and regulation of cellular respiration (1-3). Most of these responses result from interaction of NO with the heme group of the receptor guanylyl cyclase (1). A role akin to that of NO in signaling pathways has also been postulated for CO (4). CO is produced in mammals from heme by two heme oxygenases, HO-1 and HO-2 (5-8). The involvement of CO has been invoked as a factor in atherosclerosis (9), psoriasis (10), vascular constriction (11), chronic renal inflammation (12), cellular protection (13), hyperoxia-induced lung injury (14), and other physiological situations. The role of CO as an NO-like signaling molecule has received strong support from studies of heme oxygenase and nitric-oxide synthase knockouts (15), but much of the evidence, particularly that which depends heavily on inhibition of heme oxygenase by metalloporphyrins such as tin protoporphyrin IX, is tainted by ambiguities concerning the specificity of the inhibitors (16). Nevertheless, the collective evidence makes a persuasive case for at least a limited role for CO in mammalian signaling systems.Evidence has accumulated that interactions of CO and NO may influence the physiological responses to each of these agents thr...