The crystal structure of heme oxygenase-1 suggests that Asp-140 may participate in a hydrogen bonding network involving ligands coordinated to the heme iron atom. To examine this possibility, Asp-140 was mutated to an alanine, phenylalanine, histidine, leucine, or asparagine, and the properties of the purified proteins were investigated. UV-visible and resonance Raman spectroscopy indicate that the distal water ligand is lost from the iron in all the mutants except, to some extent, the D140N mutant. In the D140H mutant, the distal water ligand is replaced by the new His-140 as the sixth iron ligand, giving a bis-histidine complex. The D140A, D140H, and D140N mutants retain a trace (<3%) of biliverdin forming activity, but the D140F and D140L mutants are inactive in this respect. However, the two latter mutants retain a low ability to form verdoheme, an intermediate in the reaction sequence. All the Asp-140 mutants exhibit a new peroxidase activity. The results indicate that disruption of the distal hydrogen bonding environment by mutation of Asp-140 destabilizes the ferrous dioxygen complex and promotes conversion of the ferrous hydroperoxy intermediate obtained by reduction of the ferrous dioxygen complex to a ferryl species at the expense of its normal reaction with the porphyrin ring.Heme oxygenase (HO) 1 catalyzes the regiospecific oxidation of heme to ␣-biliverdin, CO, and free iron (1). All of the oxidative steps in the heme catabolic pathway have been extensively studied over the past 30 years, but significant gaps still exist in our understanding of this enzyme system. During its catalytic cycle, HO consumes 3 eq of O 2 and 7 reducing eq supplied by NADPH-cytochrome P450 reductase (P450 reductase) (2). The enzyme catalyzes a sequence of reactions that includes the conversion of heme to ␣-meso-hydroxyheme, ␣-meso-hydroxyheme to verdoheme, and verdoheme to ␣-biliverdin (Fig. 1). The intermediates remain bound to the enzyme throughout the catalytic cycle until ␣-biliverdin is produced and released. It is remarkable that HO can catalyze such a diverse set of reactions, because they involve the oxidation of compounds that possess different electronic and coordination properties and that have different reactivities with O 2 . This enzyme is also distinguished from all other hemoproteins in that the heme serves as the prosthetic group and substrate, and the first oxidizing species appears to be a ferric hydroperoxide (Fe(III)-OOH) rather than ferryl oxene (Fe(V)ϭO) intermediate (3). These characteristics suggest that unique interactions exist between the heme, the iron-bound O 2 , and the amino acid residues within the active site of the enzyme.In humans, HO exists in two well established forms, HO-1 and HO-2, that share moderate (ϳ45%) amino acid sequence identity but vary in their inducibility and localization (4). HO-1, also known as heat shock protein 32, is highly inducible and is the major form present in the spleen, whereas HO-2 is a constitutive enzyme that is found in highest concentration in the bra...

The human heme oxygenase-1 crystal structure suggests that Gly-139 and Gly-143 interact directly with iron-bound ligands. We have mutated Gly-139 to an alanine, leucine, phenylalanine, tryptophan, histidine, or aspartate, and Gly-143 to a leucine, lysine, histidine, or aspartate. All of these mutants bind heme, but absorption and resonance Raman spectroscopy indicate that the water coordinated to the iron atom is lost in several of the Gly-139 mutants, giving rise to mixtures of hexacoordinate and pentacoordinate ligation states. The active site perturbation is greatest when large amino acid side chains are introduced. Of the Gly-139 mutants investigated, only G139A catalyzes the NADPHcytochrome P450 reductase-dependent oxidation of heme to biliverdin, but most of them exhibit a new H 2 O 2 -dependent guaiacol peroxidation activity. The Gly-143 mutants, all of which have lost the water ligand, have no heme oxygenase or peroxidase activity. The results establish the importance of Gly-139 and Gly-143 in maintaining the appropriate environment for the heme oxygenase reaction and show that Gly-139 mutations disrupt this environment, probably by displacing the distal helix, converting heme oxygenase into a peroxidase. The principal role of the heme oxygenase active site may be to suppress the ferryl species formation responsible for peroxidase activity. Heme1 oxygenase (HO), also known as hsp32, catalyzes the NADPH-and cytochrome P450 reductase-dependent oxidation of heme to biliverdin IX␣, iron, and CO, each of which has important biological properties (1). Biliverdin is reduced by biliverdin reductase to bilirubin, which is then excreted as the glucuronic acid conjugate (2). The excretion of bilirubin is frequently impaired in newborn children due to a lag in the expression of the necessary glucuronyl transferase and in individuals with a genetic glucuronyl transferase deficiency (3). High concentrations of unconjugated bilirubin are neurotoxic, and the prevention of its accumulation through phototherapy or the inhibition of HO is of clinical importance (4 -6). However, low concentrations of biliverdin and bilirubin appear to provide important antioxidant protection (7,8). The iron released by HO is normally recycled and represents the major source of this metal in iron homeostasis, but increased iron release due to elevated heme oxygenase activity can trigger enhanced lipid and protein peroxidation (8, 9). Finally, CO appears to play a role as a signaling molecule akin to that of nitric oxide (10 -12). A role for CO in signaling pathways has received strong support from studies of HO knockout mice (13,14).The existence of two HO isoforms, HO-1 and HO-2, is well established (15-17), and a third isoform whose significance is unclear has been reported (18). HO-1 is induced by chemical agents and a variety of stress conditions, and is found in highest concentration in the spleen and liver. In contrast, HO-2 is not induced by exogenous stimuli and is found in highest concentrations in the brain and testes. The HO enzym...

Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.