Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.14.99.3 (heme oxygenase)
4,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.
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PMID:Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase. 1009 Jul 62

Heme oxygenase catalyzes the regiospecific oxidative degradation of iron protoporphyrin IX (heme) to biliverdin, CO and Fe, utilizing molecular oxygen and electrons donated from the NADPH-cytochrome P-450 reductase. The catalytic conversion of heme proceeds through two known heme derivatives, alpha-hydroxyheme and verdoheme. In order to assess the requirement of reducing equivalents in the second stage of heme degradation, from alpha-hydroxyheme to verdoheme, we have prepared the alpha-hydroxyheme complex with rat heme oxygenase isoform-1 and examined its reactivity with molecular oxygen in the absence of added electrons. Upon reaction with oxygen, the majority of the alpha-hydroxyheme in heme oxygenase is altered to a species which exhibits an optical absorption spectrum with a broad Soret band, along with the minority which is converted to verdoheme. The major product species, which is electron paramagnetic resonace-silent, can be recovered to the original alpha-hydroxyheme by addition of sodium dithionite. We have also found that oxidation of the alpha-hydroxyheme-heme oxygenase complex by ferricyanide or iridium(IV) chloride yields a species which exhibits an optical absorption spectrum and reactivity similar to those of the main product of the oxygen reaction. We infer that the oxygen reaction with the ferric alpha-hydroxyheme-heme oxygenase complex forms a ferric-porphyrin cation radical. We conclude that in the absence of reducing agents, the oxygen molecule functions mainly as an oxidant for the porphyrin ring and has no role in the oxygenation of alpha-hydroxyheme. This result corroborates our previous conclusion that the catalytic conversion of alpha-hydroxyheme to verdoheme by heme oxygenase requires one reducing equivalent along with molecular oxygen.
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PMID:Molecular oxygen oxidizes the porphyrin ring of the ferric alpha-hydroxyheme in heme oxygenase in the absence of reducing equivalent. 1040 42

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 NADPH-cytochrome 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.
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PMID:Replacement of the distal glycine 139 transforms human heme oxygenase-1 into a peroxidase. 1094 63

It has recently been suggested that, in addition to nitric oxide (NO), carbon monoxide (CO) is an important gaseous messenger which might be involved in vertebrate olfactory transduction because its effects include activation of guanylyl cyclase and the formation of cGMP. As there is no information regarding the presence of heme oxygenase-2 -- the constitutive isoform of the heme oxygenase system -- in olfactory neurons of non-rodent species, we have investigated the distribution pattern of heme oxygenase-2 in the olfactory epithelium of the bovine, a representative of macrosmatics. Localization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity of the olfactory epithelium was compared with heme oxygenase-2 and NO synthase (NOS) immunoreactivities in order to obtain possible hints at functional significance. NADPH-d activity was particularly intense in apical dendrites of receptor neurons. It was also found in Bowman glands and intraepithelial duct cells. Less intense, discrete NADPH-d activity was present also at intermediate and basal levels of the olfactory epithelium, corresponding to the layer of receptor neuron somata and basal cells. While heme oxygenase-2 activity mainly occurred in neuronal perikarya, a very intense NOS immunoreactivity, exclusively for the inducible isoform, was detected in the apical dendrites. Ultrastructurally, NADPH-d histochemistry showed distinct labelling of membranes, in particular of endoplasmic reticulum, mitochondria and nucleus. The coincident localization of the moderate NADPH-d activity and heme oxygenase-2 immunoreactivity in receptor cell perikarya suggest a functional association between NADPH-cytochrome P450 reductase and heme oxygenase-2. In contrast, dendritic localization of NADPH-d activity is topically and possibly functionally related to the presence of the inducible isoform of NOS. The results suggest that both CO and NO may be generated in bovine receptor neurons and thus involved in odorant stimulation. Based on immunocytochemical localization of synthesizing enzymes, NO might be regarded as a direct regulator of transduction related processes while CO might act as a modulator of the initial signal.
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PMID:Heme oxygenase-2 and nitric oxide synthase immunoreactivity of bovine olfactory receptor neurons and a comparison with the distribution of NADPH-diaphorase staining. 1094 53

NADPH-cytochrome P-450 reductase is the electron transfer partner for the cytochromes P-450, heme oxygenase, and squalene monooxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine has been shown to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP(+) makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently without inhibition by NADP(+). Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (k(cat)/K(m)) with NADH. Inhibition by NADP(+) was reduced by up to 4 orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2-3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P-450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P-450 2E1 Ames bacterial mutagenicity assay, the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay.
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PMID:Modification of the nucleotide cofactor-binding site of cytochrome P-450 reductase to enhance turnover with NADH in Vivo. 1238 19

Recently we have shown that ferric alpha-hydroxyhaem bound to haem oxygenase-1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196-18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric alpha-hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous alpha-hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203-213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. alpha-hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of alpha-hydroxyhaem with haem oxygenase may be small and a substantial amount of free alpha-hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air-sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH-cytochrome P450 reductase.
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PMID:The reactivity of alpha-hydroxyhaem and verdohaem bound to haem oxygenase-1 to dioxygen and sodium dithionite. 1239 55

An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 heme oxygenase gene, ho-1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The protein binds equimolar free hemin to catabolize the bound hemin to ferric-biliverdin IX alpha in the presence of oxygen and reducing equivalents, showing the heme oxygenase activity. During the reaction, verdoheme intermediate is formed with the evolution of carbon monoxide. Though both ascorbate and NADPH-cytochrome P450 reductase serve as an electron donor, the heme catabolism assisted by ascorbate is considerably slow and the reaction with NADPH-cytochrome P450 reductase is greatly retarded after the oxy-heme complex formation. The optical absorption spectra of the heme-enzyme complexes are similar to those of the known heme oxygenase complexes but have some distinct features, exhibiting the Soret band slightly blue-shifted and relatively strong CT bands of the high-spin component in the ferric form spectrum. The heme-enzyme complex shows the acid-base transition, where two alkaline species are generated. EPR of the nitrosyl heme complex has established the nitrogenous proximal ligand, presumably histidine 17 and the obtained EPR parameters are discriminated from those of the rat heme oxygenase-1 complex. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis heme oxygenase isoform-1 is different from that of rat heme oxygenase isoform-1, rather resembling that of bacterial heme oxygenase, H mu O.
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PMID:Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. Properties of the heme complex of recombinant active enzyme. 1258 Dec 8

A mouse model with liver-specific deletion of the NADPH-cytochrome P450 reductase (Cpr) gene (designated Alb-Cre/Cprlox mice) was generated and characterized in this study. Hepatic microsomal CPR expression was significantly reduced at 3 weeks and was barely detectable at 2 months of age in the Alb-Cre+/-/Cprlox+/+ (homozygous) mice, with corresponding decreases in liver microsomal cytochrome P450 (CYP) and heme oxygenase (HO) activities, in pentobarbital clearance, and in total plasma cholesterol level. Nevertheless, the homozygous mice are fertile and are normal in gross appearance and growth rate. However, at 2 months, although not at 3 weeks, the homozygotes had significant increases in liver weight, accompanied by hepatic lipidosis and other pathologic changes. Intriguingly, total microsomal CYP content was increased in the homozygotes about 2-fold at 3 weeks and about 3-fold at 2 months of age; at 2 months, there were varying degrees of induction in protein (1-5-fold) and mRNA expression (0-67-fold) for all CYPs examined. There was also an induction of HO-1 protein (nearly 9-fold) but no induction of HO-2. These data indicate the absence of significant alternative redox partners for liver microsomal CYP and HO, provide in vivo evidence for the significance of hepatic CPR-dependent enzymes in cholesterol homeostasis and systemic drug clearance, and reveal novel regulatory pathways of CYP expression associated with altered cellular homeostasis. The Alb-Cre/Cprlox mouse represents a unique model for studying the in vivo function of hepatic HO and microsomal CYP-dependent pathways in the biotransformation of endogenous and xenobiotic compounds.
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PMID:Liver-specific deletion of the NADPH-cytochrome P450 reductase gene: impact on plasma cholesterol homeostasis and the function and regulation of microsomal cytochrome P450 and heme oxygenase. 1269 46

The YLR205c gene of Saccharomyces cerevisiae does not show significant sequence identity to any known gene, except for heme oxygenase (22% to human HO-1). The YLR205 ORF was cloned and overexpressed in both Escherichia coli and S. cerevisiae. Both expression systems yielded proteins that bound heme tightly. The isolated YLR205c protein underwent reduction in the presence of either NADPH-cytochrome P450 reductase or NADH-putidaredoxin-putidaredoxin reductase but did not exhibit heme oxygenase activity. The protein exhibited modest H(2)O(2)-dependent peroxidase activities with guaiacol, potassium iodide, and 2,2(')-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS). Thus, YLR205c codes for a hemoprotein of unknown physiological function that exhibits peroxidase activity.
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PMID:Cloning and expression of a heme binding protein from the genome of Saccharomyces cerevisiae. 1269 99

Arsenic, a naturally occurring element, is present in food, soil, air and water. All human populations are exposed to arsenic and its compounds through occupational or environmental processes. Since arsenic compounds have been shown to exert their toxicity chiefly by generating reactive oxygen species, we have evaluated the effect of ascorbic acid and alpha-tocopherol on oxidative damage, antioxidant status and on xenobiotic metabolizing systems in arsenic-exposed rat liver and kidney microsomes. Arsenic exposure increases oxidative damage to lipids and proteins and decreases the levels of antioxidants and the activities of xenobiotic metabolizing enzymes. Coadministration of ascorbic acid and alpha-tocopherol to arsenic-exposed rats resulted in a reduction in the levels of lipid peroxidation, protein carbonyls and hydrogen peroxide and an elevation in the levels of reduced glutathione, ascorbic acid and alpha-tocopherol. Ascorbic acid and alpha-tocopherol treatment decreases the activity of haem oxygenase, whereas it increases the levels/ activity of cytochrome P450, cytochrome b5 and NADPH-cytochrome P450 reductase in arsenic-intoxicated rats. The results of this study provide evidence that ascorbic acid and alpha-tocopherol supplementation can improve the arsenic-induced altered microsomal functions in liver and kidney.
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PMID:Protective role of ascorbic acid and alpha-tocopherol on arsenic-induced microsomal dysfunctions. 1272 93


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