Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.99.3 (heme oxygenase)
 4,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pretreatment of rat hepatocytes with low-dose nitrogen oxide (addition of SNAP in vitro or induction of nitric oxide synthase in vitro or in vivo) imparts resistance to killing and decrease in aconitase and mitochondrial electron transfer from a second exposure to a higher dose of SNAP. Induction of this resistance is prevented by cycloheximide, indicating upregulation of protective protein(s). Ferritin levels are increased as are non-heme iron-NO EPR signals. Tin-protoporphyrin (SnPP) prevents protection, suggesting involvement of hsp32 (heme oxygenase) and/or guanylyl cyclase (GC). Cross-resistance to H2O2 killing is also observed, which is also prevented by cycloheximide and SnPP. Thus, hepatocytes possess inducible protective mechanisms against nitrogen oxide and reactive oxygen toxicity.
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PMID:Nitrogen oxide-induced autoprotection in isolated rat hepatocytes. 758 41

A truncated, soluble rat heme oxygenase-1 lacking its C-terminal, membrane-anchoring segment, and its His25-->Ala and His132-->Ala mutants have been prepared by site-directed mutagenesis and expression in Escherichia coli. We found that wild-type enzyme can degrade heme to biliverdin, but its specific activity was about one-fifth that of the native, full-length enzyme, suggesting that the C-terminal segment is important for accepting electrons from NADPH cytochrome P450 reductase. His132-->Ala mutant had an enzyme activity comparable to that of the wild-type enzyme; hence, the highly conserved His132 is not essential for the display of the heme oxygenase activity. In contrast, His25-->Ala mutation completely abolished the enzyme's catalytic activity. A five-coordinate type ferrous NO EPR spectrum was observed for the heme-heme oxygenase H25A complex. Hence, we conclude that His25 is the proximal axial ligand of the heme iron and is essential for the heme degradation activity of the enzyme.
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PMID:Demonstration that histidine 25, but not 132, is the axial heme ligand in rat heme oxygenase-1. 787 92

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.
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PMID:Rat liver heme oxygenase. High level expression of a truncated soluble form and nature of the meso-hydroxylating species. 822 46

The binding of ferrous and ferric hemes and manganese(II)- and manganese(III)-substituted hemes to heme oxygenase has been investigated by optical absorption, resonance Raman, and EPR spectroscopy. The results are consistent with the presence of a six-coordinate heme moiety ligated to an essential histidine ligand and a water molecule. The latter ionizes with a pKa approximately 8.0 to give a mixture of high-spin and low-spin six-coordinate hydroxo adducts. Addition of excess cyanide converts the heme to a hexacoordinate low-spin species. The resonance Raman spectrum of the ferrous heme-heme oxygenase complex and that of the Mn(II)protoporphyrin-heme oxygenase complex shows bands at 216 and 212 cm-1, respectively, that are assigned to the metal-histidine stretching mode. The EPR spectrum of the oxidized heme-heme oxygenase complex has a strongly axial signal with g parallel of approximately 6 and g perpendicular approximately 2. 14NO and 15NO adducts of ferrous heme-heme oxygenase exhibit EPR hyperfine splittings of approximately 20 and approximately 25 Gauss, respectively. In addition, both nitrosyl complexes show additional superhyperfine splittings of approximately 7 Gauss from spin-spin interaction with the proximal histidine nitrogen. The heme environment in the heme-heme oxygenase enzyme-substrate complex has spectroscopic properties similar to those of the heme in myoglobin. Hence, there is neither a strongly electron-donating fifth (proximal) ligand nor an electron-withdrawing network on the distal side of the heme moiety comparable to that for cytochromes P-450 and peroxidases. This observation has profound implications about the nature of the oxygen-activating process in the heme-->biliverdin reaction that are discussed in this paper.
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PMID:Resonance Raman and EPR spectroscopic studies on heme-heme oxygenase complexes. 826 Apr 99

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.
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PMID:Heme-heme oxygenase complex. Structure of the catalytic site and its implication for oxygen activation. 828 55

Heme oxygenase is a central enzyme of heme degradation and associated carbon monoxide biosynthesis. We have prepared the alpha-hydroxyheme-heme oxygenase complex, which is the first intermediate in the catalytic reaction. The active site structure of the complex was examined by optical absorption, EPR, and resonance Raman spectroscopies. In the ferric form of the enzyme complex, the heme iron is five coordinate high spin and the alpha-hydroxyheme group in the complex assumes a structure of an oxophlorin where the alpha-meso hydroxy group is deprotonated. In the ferrous form, the alpha-hydroxy group is protonated and consequently the prosthetic group assumes a porphyrin structure. The alpha-hydroxyheme group undergoes a redox-linked conversion between a keto and an enol form. The ferric alpha-hydroxyheme reacts with molecular oxygen to form a radical species. Reaction of the radical species with a reducing equivalent yields the verdoheme-heme oxygenase complex. Reaction of the ferrous alpha-hydroxyheme-heme oxygenase complex with oxygen also yields the verdoheme-enzyme complex. We conclude that the catalytic conversion of ferric alpha-hydroxyheme to verdoheme by heme oxygenase requires molecular oxygen and one reducing equivalent.
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PMID:Oxygen and one reducing equivalent are both required for the conversion of alpha-hydroxyhemin to verdoheme in heme oxygenase. 863 77

Treatment of rats with the cancer chemopreventive agent 1,2-dithiole-3-thione (D3T) resulted in a significant increase in hepatic heme oxygenase (HO) activity, which corresponded to increased protein levels of HO-1. Upon further analysis of proteins related to heme metabolism, the level of ferritin, the major iron storage protein in liver, was also found to be elevated. Diminished levels of intracellular free iron were monitored by EPR spectroscopy at times after administration of D3T that suggested that increased ferritin content sequesters intracellular iron. The increased levels of protein were associated with increased levels of steady-state RNA of HO-1 and the light (FL) and heavy (FH) subunits of ferritin. A direct relationship between enhanced rates of gene transcription and elevated levels of HO-1 and ferritin RNA was found. The inductions of FL and FH, but not HO-1, were sensitive to cycloheximide, suggesting that in vivo these genes are regulated by distinct D3T-responsive transcriptional mechanisms. The known protective roles for induced HO-1 and ferritin in cellular stress have been suggested to include increased levels of the antioxidant bilirubin and enhanced sequestration of intracellular iron into ferritin, which can effectively reduce iron-mediated reactive oxygen generation. Thus, protective actions of D3T against the cytotoxic and carcinogenic consequences of chemicals that exert electrophilic or oxidative stresses may be mediated, in part, by the induction of HO-1, FL and FH.
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PMID:Induction of hepatic heme oxygenase-1 and ferritin in rats by cancer chemopreventive dithiolethiones. 896 40

A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (DeltaHHO2) was expressed in Escherichia coli. To identify the axial heme ligand of HO-2, His-45 to Ala (DeltaH45A) and His-152 to Ala (DeltaH152A) mutants have been prepared using this expression system. DeltaH45A could form a 1:1 complex with hemin but was completely devoid of the heme degradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-DeltaH45A complex. The DeltaH152A mutant was expressed as an inclusion body and was recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 column chromatography since it eluted out of the column at the void volume. The gel filtration-purified DeltaH152A exhibited spectroscopic and enzymatic properties identical to those of wild-type. We conclude, in contrast to the previous reports (McCoubrey and Maines (1993) Arch. Biochem. Biophys. 302, 402-408; McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. (1997) J. Biol. Chem. 272, 12568-12574), that His-45, but not His-152, in heme oxygenase isoform-2 is the proximal ligand of the heme and is essential for the heme degradation activity of the enzyme. His-152 appears to play a structural role in stabilization of the heme oxygenase protein.
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PMID:Identification of histidine 45 as the axial heme iron ligand of heme oxygenase-2. 946 79

We studied the effects of nitric oxide (NO) on the control of excess cellular heme and release of catalytically active iron. Endothelial cells (ECs) exposed to hemin followed by a NO donor have a ferritin content that is 16% that of cells exposed to hemin alone. Hemin-treated ECs experience a 3.5-fold rise in non-heme, catalytic iron 2 h later, but a hemin rechallenge 20 h later results in only a 24% increase. The addition of a NO donor after the first hemin exposure prevents this adaptive response, presumably due to effects on ferritin synthesis. NO donors were found to reduce iron release from hemin, while hemin accumulated in cells. A NO donor, in a dose-dependent fashion, inhibited heme oxygenase activity, measured by bilirubin production. Using low temperature EPR spectroscopy, heme oxygenase inhibition correlated with nitrosylation of free heme in microsomes. Nitrosylation of cellular heme prevented iron release, for while there was heme oxygenase-dependent release of iron in cells incubated with hemin for 24 h, the addition of a NO donor blocked iron release. This indicates that NO readily nitrosylates intracellular free heme and prevents its degradation by heme oxygenase. Nitrosylation of heme was found to reduce sensitization of cells to oxidative injury.
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PMID:Heme and the endothelium. Effects of nitric oxide on catalytic iron and heme degradation by heme oxygenase. 972 74

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.
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PMID:Conversion of mitochondrial cytochrome b5 into a species capable of performing the efficient coupled oxidation of heme. 974 14


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