Specificity of heme oxygenase: a study with synthetic hemins

Frydman, Rosalía B.; Tomaro, Marı́a L.; Buldain, Graciela; Awruch, Josefina; Díaz, Luis Eduardo; Frydman, Benjamiǹ

doi:10.1021/bi00521a012

Cited by 69 publications

(35 citation statements)

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“…Biliverdin IXa formation from protoheme IX, as catalyzed by the enzyme heme oxygenase, requires the participation of molecular oxygen, NADPH-cytochrome c reductase, and NADPH (8,16,17,40): a reactive oxygen radical first forms an a-hydroxyheme (41,42) (44). Iron coproporphyrin III is a poor substrate (43). At this point it is not known whether a metal complex of uroporphyrin III, such as uroheme, is changed enzymatically to bactobilin.…”

Section: Methodsmentioning

confidence: 99%

Bactobilin: blue bile pigment isolated from Clostridium tetanomorphum.

Brumm¹,

Fried²,

Friedmann³

1983

Proc. Natl. Acad. Sci. U.S.A.

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A blue bile pigment, possessing four acetic and four propionic acid side chains has been isolated from extracts of the anaerobic microorganism Clostridium tetanomorphum and in smaller amounts from Propionibacterium shermanii. The compound could be prepared in larger amounts by incubation of C. tetanomorphum enzyme extracts with added 8-aminolevulinic acid. The ultraviolet-visible, infrared, and proton magnetic resonance spectra of the pigment indicate a chromophore of the biliverdin type. Field-desorption mass spectrometry of the purified methyl ester showed a strong molecular ion at m/e = 962. This corresponds to the molecular weight expected for the octamethyl ester of a bilatriene type of bile pigment structurally derived from uroporphyrin 1m or I. Of the five possible structures, two could be eliminated by proton magnetic resonance spectroscopy. The name bactobilin is proposed for this previously unreported bile pigment.The open-chain tetrapyrrole compounds known as bile pigments are widely distributed. They are found free or bound to protein in mammals, birds, amphibians, reptiles, fish, molluscs, and insects and in algae and higher plants (for reviews, see refs. 1-9). As side chains or 1&carbon ring substituents, all bile pigments described thus far have four methyl groups, two propionic acid groups, and two vinyl groups, one or both of which can be isomerized to ethylidene (4, 10, 11) or reduced to ethyl (6,10,11). These substituents are arranged in the sequence found in protoporphyrin IX; in fact, all these bile pigments are formed from protoheme (8,(12)(13)(14), whose oxidative breakdown to biliverdin is catalyzed by the enzyme heme oxygenase (15-17). Bile pigments thus far have not been detected in prokaryotes. The present paper reports the isolation and in vitro formation of a bile pigment from the bacterium Clostridium tetanomorphum. This anaerobic organism makes uroporphyrinogen, the precursor of vitamin B-12, but not heme, protoporphyrinogen, or coproporphyrinogen. The isolated bile pigment is of interest not only because of its detection in a prokaryote, but also because its 13-carbon ring substituents, four acetic acid and four propionic acid groups, correspond to those of a uroporphyrin and not of protoporphyrin. MATERIALS AND METHODSSupplies. -Aminolevulinic acid was obtained from Sigma;biliverdin IXa dimethyl ester and the fully esterified methyl esters of uroporphyrin III, coproporphyrin III, protoporphyrin IX, and heptacarboxylporphyrin I, were from Porphyrin Products (Logan, UT). The n-hexane used for flash chromatography was "95 + %" (Aldrich), whereas the n-hexane for thin-layer chromatography was 99 mol % pure (Fisher). Other chemicals used were reagent grade or better. Silica gel for flash chromatography and a flash chromatography column, 3.4-cm diameter, were obtained from J. T. Baker. Whatman high-performance silica thin-layer chromatography plates with a preadsorbent spotting area (type LHP-K, 10 x 20 cm) and E. Merck precoated cellulose thin-layer chromatography plates were...

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Section: Methodsmentioning

confidence: 99%

Bactobilin: blue bile pigment isolated from Clostridium tetanomorphum.

Brumm¹,

Fried²,

Friedmann³

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…In particular, the crystal structure of the heme-bound form (heme-HO-1 complex) revealed that the highly conserved Lys 179 and Arg 183 residues interact electrostatically with the heme propionate groups and that these residues play important roles in the ␣-regiospecificity of HO catalysis (22,23,26). The necessity of the heme propionate side chains at the C-6 and C-7 positions for the HO reaction has been also suggested from previous studies on the substrate specificity of HO using a variety of modified hemes (27,28). Specifically, a modified heme, in which the C-6 and C-7 propionates are substituted with butyrates, can be degraded to bilirubin at only half the rate observed with the normal heme under the usual HO reaction conditions containing CPR and biliverdin reductase, whereas the heme modified with acetates cannot serve as a substrate for HO.…”

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confidence: 76%

The Reactions of Heme- and Verdoheme-Heme Oxygenase-1 Complexes with FMN-depleted NADPH-cytochrome P450 Reductase

Higashimoto¹,

Satō²,

Sakamoto³

et al. 2006

J. Biol. Chem.

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Electrons utilized in the heme oxygenase (HO) reaction are provided by NADPH-cytochrome P450 reductase (CPR). To investigate the electron transfer pathway from CPR to HO, we examined the reactions of heme and verdoheme, the second intermediate in the heme degradation, complexed with rat HO-1 (rHO-1) using a rat FMN-depleted CPR; the FMN-depleted CPR was prepared by dialyzing the CPR mutant, Y140A/ Y178A, against 2 M KBr. Degradation of heme in complex with rHO-1 did not occur with FMN-depleted CPR, notwithstanding that the FMN-depleted CPR was able to associate with the heme-rHO-1 complex with a binding affinity comparable with that of the wild-type CPR. Thus, the first electron to reduce the ferric iron of heme complexed with rHO-1 must be transferred from FMN. In contrast, verdoheme was converted to the ferric biliverdin-iron chelate with FMN-depleted CPR, and this conversion was inhibited by ferricyanide, indicating that electrons are certainly required for conversion of verdoheme to a ferric biliverdin-iron chelate and that they can be supplied from the FMN-depleted CPR through a pathway not involving FMN, probably via FAD. This conclusion was supported by the observation that verdoheme dimethyl esters were accumulated in the reaction of the ferriprotoporphyrin IX dimethyl ester-rHO-1 complex with the wild-type CPR. Ferric biliverdin-iron chelate, generated with the FMN-depleted CPR, was converted to biliverdin by the addition of the wild-type CPR or desferrioxamine.Thus, the final electron for reducing ferric biliverdin-iron chelate to release ferrous iron and biliverdin is apparently provided by the FMN of CPR.Heme oxygenase (HO 4 ; EC 1.14.99.3) is a microsomal enzyme that catalyzes the degradation of heme to biliverdin IX␣, carbon monoxide (CO), and free iron in the presence of NADPH-cytochrome P450 reductase (CPR; EC 1.6.2.4) (1-3). The HO reaction proceeds via a multistep mechanism as shown in Fig. 1 (4, 5). The first step is the oxidation of heme to ␣-hydroxyheme, requiring molecular oxygen (O 2 ) and reducing equivalents supplied by CPR (6 -8). The second step is the formation of verdoheme with the concomitant release of hydroxylated ␣-meso carbon as CO (9 -11). The third step is the conversion of ␣-verdoheme to a biliverdin-iron chelate, for which O 2 and electrons from CPR are also required (12). In the last step, the iron of the biliverdin-iron chelate is reduced, and finally ferrous iron and biliverdin are released from HO. The conversion of verdoheme to the ferric biliverdin-iron chelate along with the release of biliverdin is a rate-limiting step in the HO reaction (13). The mechanism of degradation of verdoheme is the least understood of the three successive oxygenation reactions during the HO catalysis, although some chemical mechanisms have been proposed (12,14).CPR is an FAD-and FMN-containing protein that supplies electrons required for the HO reaction as well as for a variety of other hemoproteins, such as cytochrome P450s and cytochrome b 5 . With the latter hemoproteins, it is thought t...

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“…The reduction of all the hematobiliverdin isomers t o hematobilirubins by biliverdin reductase also falls in line with the substrate specificity found for biliverdin reductase with other biliverdin isomers [4, 15, 161. The hematobiliverdin IXP was also found to be reduced at higher rates by molecular form 3 of biliverdin reductase, as was the case with biliverdin IXg [15] and biliverdin XIIIB [16], very likely as a result of a conformational feature of the P isomers, which is currently under study.…”

Section: Discussionmentioning

confidence: 99%

“…Biliverdins IXa and IXP were obtained by the chemical oxidation of hemin IX (purchased from Sigma Chemical Co.) and separated from the other two biliverdin IX isomers as [2] and was crystallized from chloroform/hexane. NADPH, NADH, 3-acetyl-NADP+, 3-acetyl-NADH, deamino-NADPH, deamino-NADH, glucose 6-phosphate and glucose-6-phosphate dehydrogenase were purchased from Sigma Chemical Co. Silica-gel-coated thinlayer chromatography (TLC) plates were purchased from Merck (0.25 mm layer thickness).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The chemical and enzymatic oxidation of hematohemin IX. Identification of hematobiliverdin IXalpha as the sole product of the enzymatic oxidation

et al. 1986

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Oxidative cleavage of hematohemin IX in pyridine solution in the presence of ascorbic acid (coupled oxidation), followed by esterification of the products with boron trifluoride/methanol produced the four possible hematobiliverdin dimethyl esters in 11.1 YO overall yield. Transetherifications took place simultaneously with the esterification reaction and resulted in the formation of the dimethyl ester of hematobiliverdin IXy 8 a,l3 a-dimethyl ether (1.8%), the dimethyl ester of hematobiliverdin IXP 13 a,lSa-dimethyl ether (1.9"/), the dimethyl ester of hematobiliverdin 1x6 8 a-monomethyl ether (1.4%), and the dimethyl ester of hematobiliverdin IXa 18 a-monomethyl ether (0.4%). The latter was the sole product obtained after the enzymatic oxidation of hematohemin with heme oxygenase, after esterification of the reaction product with boron trifluoride/methanol. When the esterification step was omitted hematobiliverdin IXa was obtained from the enzymatic oxidation. The structures of the hematobiliverdin derivatives were secured by their NMR and mass spectra data. Saponification of the dimethyl esters afforded the hematobiliverdin methyl ethers, which were excellent substrates of biliverdin reductase and were readily reduced to the corresponding bilirubins. Hematobiliverdin IXa was also a good substrate of biliverdin reductase. It is concluded that the enzymatic oxidation of hematohemin IX by heme oxygenase is a-selective, while biliverdin reductase shows no selectivity in the reduction of the four hematobiliverdin isomers.Heme IX (or b) is degraded in the general metabolism by a microsomal heme oxygenase, which selectively cleaves the a-meso carbon of the hemin to give biliverdin IX a and the latter is then reduced in mammals by a cytosolic biliverdin reductase to bilirubin IXa. We have extensively studied the substrate specificity of both enzymes and we established, by making use of a large number of synthetic hemins and bi-

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Specificity of heme oxygenase: a study with synthetic hemins

Cited by 69 publications

References 19 publications

Bactobilin: blue bile pigment isolated from Clostridium tetanomorphum.

Bactobilin: blue bile pigment isolated from Clostridium tetanomorphum.

The Reactions of Heme- and Verdoheme-Heme Oxygenase-1 Complexes with FMN-depleted NADPH-cytochrome P450 Reductase

The chemical and enzymatic oxidation of hematohemin IX. Identification of hematobiliverdin IXalpha as the sole product of the enzymatic oxidation

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