Heme-heme Oxygenase Complex Research Articles

Inactivation of the Heme Degrading Enzyme IsdI by an Active Site Substitution That Diminishes Heme Ruffling

IsdG and IsdI are paralogous heme degrading enzymes from the bacterium Staphylococcus aureus. Heme bound by these enzymes is extensively ruffled such that the meso-carbons at the sites of oxidation are distorted toward bound oxygen. In contrast, the canonical heme oxygenase family degrades heme that is bound with minimal distortion. Trp-66 is a conserved heme pocket residue in IsdI implicated in heme ruffling. IsdI variants with Trp-66 replaced with residues having less bulky aromatic and alkyl side chains were characterized with respect to catalytic activity, heme ruffling, and electrochemical properties. The heme degradation activity of the W66Y and W66F variants was approximately half that of the wild-type enzyme, whereas the W66L and W66A variants were inactive. A crystal structure and NMR spectroscopic analysis of the W66Y variant reveals that heme binds to this enzyme with less heme ruffling than observed for wild-type IsdI. The reduction potential of this variant (-96 ± 7 mV versus standard hydrogen electrode) is similar to that of wild-type IsdI (-89 ± 7 mV), so we attribute the diminished activity of this variant to the diminished heme ruffling observed for heme bound to this enzyme and conclude that Trp-66 is required for optimal catalytic activity.

Reactions of meso-hydroxyhemes with carbon monoxide and reducing agents in search of the elusive species responsible for the g = 2.006 resonance of carbon monoxide-treated heme oxygenase. Isolation of diamagnetic iron(II) complexes of octaethyl-meso-hydroxyporphyrin.

To examine possible models for the g = 2.006 resonance seen when the hydroxylated heme-heme oxygenase complex in the Fe(III) state is treated with CO, the reactivities of CO and reducing agents with (py)(2)Fe(III)(OEPO) and [Fe(III)(OEPO)](2) (OEPO is the trianion of octaethyl-meso-hydroxyporphyrin) have been examined. A pyridine solution of (py)(2)Fe(III)(OEPO) reacts in a matter of minutes with zinc amalgam (or with hydrazine) under an atmosphere of dioxygen-free dinitrogen to produce bright-red (py)(2)Fe(II)(OEPOH).2py.0.33H(2)O, which has been isolated in crystalline form. The (1)H NMR spectrum of (py)(2)Fe(II)(OEPOH) in a pyridine-d(5) solution is indicative of the presence of a diamagnetic compound, and no EPR resonance was observed for this compound. Treatment of a solution of (py)(2)Fe(II)(OEPOH) in pyridine-d(5) with carbon monoxide produces spectral changes after a 30 s exposure that are indicative of the formation of diamagnetic (OC)(py)Fe(II)(OEPOH). Treatment of a green pyridine solution of (py)(2)Fe(III)(OEPO) with carbon monoxide reveals a slow color change to deep red over a 16 h period. Although a resonance at g = 2.006 was observed in the EPR spectrum of the sample during the reaction, the isolated product is EPR silent. The spectroscopic features of the final solution are identical to those of a solution formed by treating (py)(2)Fe(II)(OEPOH) with carbon monoxide. Addition of hydrazine to solutions of (OC)(py)Fe(II)(OEPOH) produces red, diamagnetic (OC)(N(2)H(4))Fe(II)(OEPOH).py in crystalline form. The X-ray crystal structures of (py)(2)Fe(II)(OEPOH).2py.0.33H(2)O and (OC)(N(2)H(4))Fe(II)(OEPOH).py have been determined. Solutions of diamagnetic (OC)(N(2)H(4))Fe(II)(OEPOH).py and (OC)(py)Fe(II)(OEPOH) are extremely air sensitive and are immediately converted in a pyridine solution into paramagnetic (py)(2)Fe(III)(OEPO) in the presence of dioxygen.

Conversion of mitochondrial cytochrome b5 into a species capable of performing the efficient coupled oxidation of heme.

Histidine-63, one of the heme axial ligands in outer mitochondrial membrane cytochrome b5 (OM cyt b5) has been replaced by a methionine. The H63M variant performs the efficient and regioselective coupled oxidation of heme in order to produce >90% of the alpha-isomer of verdoheme. The variant was characterized by electronic, EPR, and NMR spectroscopic studies which indicate that the ferric form is a high-spin species whose heme is coordinated by histidine-39 in the proximal site and likely by water in the distal site. The coordination of methionine to the ferric heme was ruled out on the basis of NMR spectroscopic studies. Addition of imidazole to a solution of the ferric variant results in the formation of a species axially coordinated by imidazole and histidine-63. The reduction potential of the variant was found to be +110 mV in the absence of exogenous imidazole and -92 mV in the presence of imidazole. These values compare well with the reduction potential of myoglobin (50 mV) and wild-type OM cyt b5 (-102 mV), respectively, consistent with the axial ligation described above. The ferrous variant, on the other hand, is a low-spin species coordinated by histidine-39 and methionine-63. Carbon monoxide (CO) readily displaces Met-63 from its coordination site on the ferrous heme, whereas CO cannot completely displace Met-63 from its coordination site on verdoheme. Consequently, the mechanism of inhibition for the oxidation of verdoheme to iron-biliverdin in the H63M variant appears to be similar to that observed for the heme-heme oxygenase complex in the presence of CO.

Heme oxygenase active-site residues identified by heme-protein cross-linking during reduction of CBrCl3.

The reduction of CBrCl3 by the heme-heme oxygenase complex forms dissociable and covalently bound heme products. No such products are formed with mesoheme in which the heme vinyl substituents are replaced by ethyl groups. The dissociable heme products are chromatographically similar but not identical to those obtained in the analogous reaction with myoglobin. Tryptic digestion of the heme-protein adduct and Edman sequencing and mass spectrometric analysis of the heme-linked peptide identify His-25, the proximal iron ligand, as the alkylated residue. Reaction of CBrCl3 with the heme complexes of the T135V mutant and a Delta221 C-terminal truncated protein yields heme-linked peptides in addition to that from the wild-type reaction. The sequence of the principal labeled peptide from the T135V reaction, 205TAFLLNIQLFEELQELLTHDTK226 , and the lability of the adduct suggest the heme is attached to one of the carboxylic acid residues. A carboxylic acid residue is probably also labeled in the modified peptide 49LVMASLYHIYVALEEEIER67 from the Delta221 truncated protein. Thus, addition of the reductively generated trichloromethyl radical to a heme vinyl group produces a species that alkylates active-site residues. The changes in the alkylated residue caused by the Thr-135 mutation or truncation of the protein places residues in the sequences 49-67 and 205-226 within the active site. Furthermore, this is the first demonstration that heme oxygenase, like cytochrome P450, may catalyze the reductive metabolism of halocarbons and thus contribute to the toxicity of these agents.

Ligation of the iron in the heme-heme oxygenase complex: X-ray absorption, electronic absorption and magnetic circular dichroism studies

Heme oxygenase (HO) catalyzes the first steps in the breakdown of heme to biliverdin and carbon monoxide. It is a membrane-bound protein that has been shown to exist in two isoforms, HO-1 and HO-2. Recently, a soluble, truncated form of rat HO-1 (rHO) lacking the 23 amino-acid membrane anchor has been expressed in E. coli. Extended X-ray absorption fine structure (EXAFS) data on ferric rHO and its fluoride derivative support assignment of the axial iron ligands as oxygen and/or nitrogen donors having distances similar to ferric myoglobin. The electronic absorption and magnetic circular dichroism (MCD) spectra of the ferric and ferrous protoheme complexes of rHO as well as various ligand adducts are very similar to the corresponding spectra of myoglobin. The present study is the first investigation of the heme-heme oxygenase complex with EXAFS and MCD spectroscopy and establishes that the proximal ligand to the heme in rHO is histidine. Furthermore, the close similarity between the electronic absorption and MCD spectra of ferric rHO and myoglobin over the pH range 6 to 10 is consistent with distal heme ligation of ferric rHO as a water molecule or hydroxide ion, depending on pH. Taken together and in conjunction with the results of earlier studies, EXAFS, electronic absorption, and MCD spectroscopy solidly establish that the ligands to the heme in rHO are identical to those in myoglobin, namely, histidine/H 2O at low pH and histidine/OH at high pH.

Cloning and expression of cDNA for soluble form of rat heme oxygenase-1.

Heme oxygenase catalyzes the oxidation of heme to biliverdin and carbon monoxide. The gene encoding the truncated soluble rat heme oxygenase-1 (Metl-Pro267) was cloned. The enzyme protein was expressed in E. coli JM109 and purified to homogeneity. The molecular weight of the recombinant enzyme was 30 kDa as assessed by SDS-polyacrylamide gel electrophoresis. From a 3-L culture, about 90 mg of the purified enzyme was routinely obtained. The dependency of the heme oxygenase reaction catalyzed by the soluble enzyme on the NADPH-cytochrome P-450 reductase concentrations and the effect of catalase on the reaction were examined to compare with the purified membrane-bound form of heme oxygenase-1 (Yoshida and Kikuchi, 1978b). The activity of the soluble enzyme was inhibited at high concentrations of NADPH-cytochrome P-450 reductase and the inhibition was not alleviated by addition of catalase unlike the membrane-bound form. The ferric iron of the heme-heme oxygenase complex was in a typical high spin state at acidic to neutral pH (pH 6.5-7.0) but conversion to low spin state was observed at basic pH (pH 9-10). The heme bound to heme oxygenase was converted to biliverdin at a stoichiometric ratio of unity in the presence of NADPH-cytochrome P-450 reductase system. During the heme degradation of the heme-heme oxygenase complex under atmospheric oxygen, several intermediates, that is, oxygenated heme and verdoheme, were spectrally discriminated.

Oxygen-Bound Heme-Heme Oxygenase Complex: Evidence for a Highly Bent Structure of the Coordinated Oxygen

Oxygen-Bound Heme-Heme Oxygenase Complex: Evidence for a Highly Bent Structure of the Coordinated Oxygen

Heme oxygenase (HO-1). Evidence for electrophilic oxygen addition to the porphyrin ring in the formation of alpha-meso-hydroxyheme.

Previous studies have established that reaction of the rat heme-heme oxygenase complex with H2O2 proceeds normally to give verdoheme, whereas reaction of the complex with meta-chloroperbenzoic acid yields a ferryl (FeIV = O) species and a protein radical but no verdoheme. The heme-heme oxygenase complex is shown here to react regiospecifically with ethyl hydroperoxide to give alpha-meso-ethoxyheme. Formation of this product exactly parallels the formation of alpha-meso-hydroxyheme in the normal reaction supported by cytochrome P450 reductase/NADPH or H2O2. These results rule out a nucleophilic mechanism for the alpha-meso-hydroxylation catalyzed by heme oxygenase and indicate that it involves electrophilic (or possibly radical) addition of the distal oxygen of iron-bound peroxide (FeIII-OOH) to the porphyrin ring.

Heme-heme oxygenase complex: structure and properties of the catalytic site from resonance Raman scattering.

The resonance Raman spectra of ferric and ferrous forms of the heme-heme oxygenase (HO) complex (isoform 1) clarify several structural features of the catalytic active site. Isotopic substitution studies of the central iron atom of the heme demonstrate that the line at 218 cm-1 in the ferrous ligand-free form of the complex originates from the iron-histidine stretching mode. The presence of a Raman line at this frequency confirms that the fifth ligand coordinating to the heme is a neutral imidazole from a histidine residue. The modes associated with CO in the carboxy derivative of the ferrous enzyme complex have typical frequencies of histidine-bound heme proteins such as myoglobin. In the ferric form of the complex, at alkaline pH, hydroxide is identified as the bound exogenous ligand, and at neutral pH we infer that water is bound. Thus, the coordination of the heme-HO complex is the same as that in myoglobin. However, in a comparison of the low-frequency vibrational modes in the resonance Raman spectrum of the heme-HO complex to those of myoglobin, the spectra are found to be very different, indicating that the interactions between the heme and its amino acid pocket in these two proteins are quite different. The neutral imidazole may play several important roles in the physiological function of the heme-HO complex.

Heme-heme oxygenase complex. Structure of the catalytic site and its implication for oxygen activation.

Heme oxygenase, a central monooxygenase enzyme of the heme catabolism and the associated generation of carbon monoxide, forms a 1:1 stoichiometric complex with iron protoporphyrin IX, which is a prosthetic active center and at the same time the substrate of the enzyme. By using EPR, resonance Raman, and optical absorption spectroscopic techniques, we have determined the axial ligand coordination of the enzyme-heme complex. The ferric heme iron in the heme-enzyme complex at neutral pH is six-coordinate high spin, while at alkaline pH (pKa 7.6), the complex becomes low spin. Spectra of ferrous forms of the complex indicate that histidine serves as the iron proximal axial ligand and that the residue is in its neutral imidazole rather than its imidazolate protonation state. Thus, the active site of the heme-heme oxygenase complex has a myoglobin-like structure rather than an active site similar to the large cytochrome P-450 class of monooxygenases. As a consequence, the activated form of the heme-heme oxygenase complex, a peroxo intermediate, is different from that of the cytochrome P-450 monooxygenases, in which the activated form is an oxo intermediate. The overall catalytic mechanism is probably more closely related to that of other monooxygenases with myoglobin-like active sites, such as secondary amine monooxygenase.

Rat liver heme oxygenase. High level expression of a truncated soluble form and nature of the meso-hydroxylating species.

A rat heme oxygenase (HO-1) gene without the sequence coding for the last 23 amino acids has been constructed and expressed behind the pho A promoter in Escherichia coli. The enzyme is expressed at high levels as a soluble catalytically active protein that causes the bacterial cells to accumulate biliverdin. The purified truncated heme-heme oxygenase complex is spectroscopically indistinguishable from the complex with the native enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. Reaction of the recombinant heme-heme oxygenase complex with H2O2 produces a species with the spectroscopic properties of verdoheme. Unidentified products are obtained when this intermediate is directly extracted from the protein, but biliverdin is obtained if the verdoheme-protein complex is exposed to cytochrome P450 reductase and NADPH before the extraction step. In contrast, reaction of the heme-heme oxygenase complex with meta-chloroperbenzoic acid (mCPBA), tert-butylhydroperoxide, or cumene hydroperoxide yields a ferryl (FeIV = O) complex. Reaction of the heme-heme oxygenase complex with mCPBA also produces an EPR-detectable protein radical. In accord with formation of a ferryl intermediate, recombinant heme oxygenase catalyzes the mCPBA- and alkylhydroperoxide-dependent peroxidation of 2-methoxyphenol (guaiacol). Guaiacol oxidation is not observed during turnover of the enzyme by cytochrome P450 reductase/NADPH or H2O2. Conversely, biliverdin is not formed with tert-butylhydroperoxide or mCPBA. H2O2 thus supports the first step of the normal catalytic oxidation of heme by heme oxygenase, but alkyl and acyl hydroperoxides do not. These results suggest that the alpha-meso-hydroxylation required for biliverdin formation is mediated by the distal of the two oxygens in the iron-dioxygen intermediate (Fe-O-O) engendered by reaction with either cytochrome P450 reductase/NADPH or H2O2.

The step of carbon monoxide liberation in the sequence of heme degradation catalyzed by the reconstituted microsomal heme oxygenase system.

Oxidative degradation of protoheme to biliverdin IXa catalyzed by the microsomal heme oxygenase system proceeds in the following sequence: protoheme, a-hydroxyheme, the 688-nm substance, a biliverdin-iron complex, and biliverdin. In the heme oxygenase reaction, the a-methene carbon of heme is liberated as carbon monoxide. The 688-nm substance, a newly found intermediate (Yoshida, T., Noguchi, M., and Kikuchi G. (1980) J. Biochem (Tokyo) 88, 557-563), has an extremely high affinity for carbon monoxide and, therefore, when the heme-heme oxygenase complex was incubated with the NADPH-cytochrome P-450 reductase system under a gas mixture of air and CO at a volumetric ratio of 5:1, about 80% of the heme was found to stay at the level of the CO-reduced form of the 688-nm substance. Taking advantage of these properties of the 688-nm substance, an attempt was made to determine whether carbon monoxide is liberated before or after the step of the 688-nm substance, by using 14C-labeled heme synthesued from [5-‘4C]G-aminolevulinic acid. The complex of 14C-heme and heme oxygenase was incubated with the NADPH-cytochrome P-450 reductase system in either air or a gas mixture of air and CO, and the content of 14C0 in the gas phase was analyzed by means of converting 14C0 into 14C02 by oxidation. In either reaction system, practically equal amounts of 14C02 were obtained; the radioactivities recovered in COz were 11 to 12% of the original radioactivity of 14C-heme used. The values of 11 to 12% are very close to the theoretical value of 12.5% (or one-eighth) which is expected to be recovered in C 0 2 when all the I4C-heme was completely degraded to biliverdin. These findings clearly indicate that carbon monoxide is liberated at the step of the formation of 688-nm substance from a-hydroxyheme and, hence, the 688-nm substance evidently lacks the a-methene carbon.