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PubMed2004

The source of heme for vascular heme oxygenase II: de novo heme biosynthesis in rat aorta.

Heme is an essential prosthetic group or substrate for many proteins, including hemoglobin, and hemo enzymes such as nitric oxide synthase, soluble guanylyl cyclase, and heme oxygenase (HO). HO is responsible for the breakdown of heme into equimolar amounts of biliverdin, iron, and carbon monoxide, the latter of which is thought to play a role in the regulation of vascular tone. It is not clear whether the source of heme for cardiovascular functions is derived from uptake from the extracellular milieu or synthesis. In this study, we tested the hypothesis that blood vessels obtain their supply of heme for HO through de novo synthesis. Adult male Sprague-Dawley rat aorta was incubated at 37 degrees C in Krebs' solution with 1 micro M [14C]delta-aminolevulinic acid (ALA). [14C]ALA uptake was linear for about 30 min and reached a plateau at approximately 100 min. The radioactivity was incorporated into porphyrins and heme as determined by esterification of 14C-labelled metabolites and thin-layer chromatography. The first and rate-limiting step of heme biosynthesis is catalyzed by ALA synthase (ALA-S), the activity of which was determined in rat aorta using a radiometric assay, approximately 250 nmol x (g wet mass)(-1) x h(-1). Inducing HO-1 in rat aorta with S-nitroso-N-acetylpenicillamine (500 micro M) did not increase ALA-S activity as compared with basal activity levels of the enzyme. It appears that there is a sufficient amount of heme available under basal ALA-S activity conditions to meet the increased demand for heme resulting from HO-1 induction. These observations indicate that the complete enzymatic pathway for de novo heme biosynthesis resides in rat aorta and furthermore indicate that de novo heme synthesis is capable of supplying a substantial portion of the heme substrate for HO in the aorta.

5-Aminolevulinate Synthetase↗
PubMed1998

Neisseria gonorrhoeae heme biosynthetic mutants utilize heme and hemoglobin as a heme source but fail to grow within epithelial cells.

Many bacterial pathogens, including pathogenic neisseriae, can use heme as an iron source for growth. To study heme utilization by Neisseria gonorrhoeae, two heme biosynthetic mutants were constructed, one with a mutation in hemH (the gene encoding ferrochelatase) and one with a mutation in hemA (the gene encoding gamma-glutamyl tRNA reductase). The hemH mutant failed to grow without an exogenous supply of heme or hemoglobin, whereas the hemA mutant failed to grow unless heme, hemoglobin, or heme precursors were present. Growth of the mutants with hemoglobin required expression of the hemoglobin receptor (HpuAB) and was TonB dependent. However, growth with heme required neither HpuAB nor TonB. An fbpA mutant grew normally when either heme or hemoglobin was present in the medium. The heme biosynthetic mutants showed reduced intracellular survival, compared to the parent strain, within A-431 endocervical epithelial cell cultures. These studies demonstrate that in addition to synthesizing their own heme, N. gonorrhoeae strains are able to internalize and utilize exogenous heme independently of FbpA but appear unable to obtain heme from within epithelial cells for growth.

Aldehyde Oxidoreductases↗
PubMed1995

Expression of the mRNA of heme-binding protein 23 is coordinated with that of heme oxygenase-1 by heme and heavy metals in primary rat hepatocytes and hepatoma cells.

A 23-kDa protein with high affinity for heme (KD = 55 nM), therefore termed heme-binding protein 23 kDa (HBP23), was purified from rat liver cytosol [Iwahara, S., et al. (1995) Biochemistry 34, 13398-13406]. Homology search of the cloned HBP23 cDNA revealed that this protein belongs to a recently recognized class of thiol peroxidases, the antioxidant peroxiredoxin family. Since HBP23 gene expression was highest in the liver, HBP23 mRNA regulation by heme and heavy metals was investigated in cultures of primary rat hepatocytes and mouse hepatoma Hepa 1-6 cells. In both cell cultures HBP23 mRNA levels were upregulated in a time- and dose-dependent manner by heme. Heme-dependent induction of HBP23 mRNA occurred coordinately with that of the heme-metabolizing enzyme heme oxygenase-1, which was recently identified as inducible by oxidative stress. Treatment of primary rat hepatocyte or hepatoma cell cultures with the heavy metals CdCl2 (10 microM) and CoCl2 (300 microM) induced in parallel HBP23 and HO-1 mRNA levels, in the case of CdCl2 to even higher levels than heme. By contrast, mRNA expression of another heme binding protein, hemopexin, was not induced in hepatocyte cell cultures by heme or heavy metals. The data suggest that the expression of HBP23 and HO-1 mRNA is regulated by (a) similar mechanism(s) in liver and that both genes could play a common physiological role as antioxidants and/or in heme metabolism.

Animals↗
PubMed2005

Analysis of a heme-dependent signal transduction system in Corynebacterium diphtheriae: deletion of the chrAS genes results in heme sensitivity and diminished heme-dependent activation of the hmuO promoter.

The Corynebacterium diphtheriae hmuO gene encodes a heme oxygenase that is involved in the utilization of heme as an iron source. Transcription of hmuO is activated by heme or hemoglobin and repressed by iron and DtxR. Previous studies with Escherichia coli showed that heme-dependent transcriptional activation of an hmuO promoter-lacZ fusion was dependent on the cloned C. diphtheriae chrA and chrS genes (chrAS), which encode the response regulator and sensor kinase, respectively, of a two-component signal transduction system. In this study, nonpolar deletions in the chrAS genes were constructed on the chromosome of C. diphtheriae. Mutations in chrAS resulted in marked reduction in heme-dependent transcription of hmuO, which indicates that the ChrA/S system is a key regulator at the hmuO promoter. However, low but significant levels of heme-specific transcriptional activity were observed at the hmuO promoter in the chrAS mutants, suggesting that an additional heme-dependent activator is involved in hmuO expression. The chrAS mutants were also sensitive to heme, which was observed only in stationary-phase cultures and correlated with reduced cell viability. The heme sensitivity of the mutants was not due to reduced expression of hmuO, and these results suggest that additional factors controlled by the ChrA/S system may be involved in protection against heme toxicity. Transcriptional analysis of the chrAS operon revealed that it was not autoregulated or affected by iron or heme levels.

Bacterial Proteins↗
PubMed2006

Role of heme-protein covalent bonds in mammalian peroxidases. Protection of the heme by a single engineered heme-protein link in horseradish peroxidase.

Oxidation of SCN-, Br-, and Cl- (X-) by horseradish peroxidase (HRP) and other plant and fungal peroxidases results in the addition of HOX to the heme vinyl group. This reaction is not observed with lactoperoxidase (LPO), in which the heme is covalently bound to the protein via two ester bonds between carboxylic side chains and heme methyl groups. To test the hypothesis that the heme of LPO and other mammalian peroxidases is protected from vinyl group modification by the hemeprotein covalent bonds, we prepared the F41E mutant of HRP in which the heme is attached to the protein via a covalent bond between Glu41 and the heme 3-methyl. We also examined the E375D mutant of LPO in which only one of the two normal covalent heme links is retained. The prosthetic heme groups of F41E HRP and E375D LPO are essentially not modified by the HOBr produced by these enzymes. The double E375D/D225E mutant of LPO that can form no covalent bonds is inactive and could not be examined. These results unambiguously demonstrate that a single heme-protein link is sufficient to protect the heme from vinyl group modification even in a protein (HRP) that is normally highly susceptible to this reaction. The results directly establish that one function of the covalent heme-protein bonds in mammalian peroxidases is to protect their prosthetic group from their highly reactive metabolic products.

Animals↗
PubMed2004

The source of heme for vascular heme oxygenase I: heme uptake in rat aorta.

During the last decade, heme oxygenase (HO) and carbon monoxide (CO) have garnered substantial research interest in terms of cell and organ regulation, especially as they bear on the central nervous system, organ transplantation, and the cardiovascular system. While the enzymatic mechanism, substrates, and products of HO are well known, it is not clear whether the cardiovascular system derives its supply of the heme substrate through de novo synthesis or uptake from the extracellular milieu. The objective of the present study was to test the latter possibility in rat aorta and to determine the influence of plasma proteins that bind heme in vivo, viz. hemopexin and albumin. Aortic tissue was exposed to [14C]heme in vitro, and the concentration and time dependence of heme uptake was assessed. The presence of hemopexin or albumin in the incubation medium dramatically decreased heme uptake by the aorta. Heme uptake by aortic tissue was not altered after induction of HO-1, which would be expected to increase tissue heme demand. In summary, the rat, isolated aorta was capable of obtaining heme from its external milieu, but this was obtunded in the presence of the plasma proteins hemopexin or albumin. For normal physiological situations, heme uptake may not be a usual source of substrate for vascular HO and hemoenzymes such as nitric oxide synthase, soluble guanylyl cyclase, and cyclooxygenase.

Animals↗
PubMed1998

Heme and the endothelium. Effects of nitric oxide on catalytic iron and heme degradation by heme oxygenase.

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.

Adaptation, Physiological↗
PubMed1997

Kinetics of heme interaction with heme-binding proteins: the effect of heme aggregation state.

The kinetics of the interaction of heme with hemopexin and albumin was monitored by measuring the time dependence of changes in the Soret absorption spectra. Since the protein binding sites can only bind heme monomers, the binding kinetics apparently reflected the slow dissociation of heme dimers, resulting from dimer/monomer equilibria in aqueous heme solutions. The dissociation of heme dimers is characterized by the rate constant of (3-4) x 10(-3) s(-1). The measurements further revealed significant differences in the kinetic profiles (slowing down the binding interaction) that were dependent on the storage time of heme solutions at room temperature. These presumably responded to the gradual formation of higher aggregates of heme, which cannot dissociate into dimers/monomers. Alternatively, partial autooxidation of heme molecules could increase the stability of heme dimers and obstruct specific binding of heme to the proteins.

Binding Sites↗
PubMed2002

An examination of heme action in gene expression: heme and heme deficiency affect the expression of diverse genes in erythroid k562 and neuronal PC12 cells.

The aim of this study was to identify key genes whose expression is altered by heme and heme deficiency in the human erythroleukemia K562 cells and in the NGF-induced rat pheochromocytoma neuronal PC12 cells, respectively. By quantitative RT-PCR, Northern blotting, and Western blotting analyses, we found that the expression of the CDK inhibitors p18 and p21 was upregulated at the early and late stages of heme-induced erythroid differentiation of K562 cells, respectively, while the expression of cyclin D1 was downregulated. Data from succinyl acetone and desferrioxamine treatments suggest that these effects of heme in K562 cells were specific. Further, by microarray expression analysis, we found that inhibition of heme synthesis by succinyl acetone in NGF-induced PC12 cells drastically altered the expression of several groups of important neuronal genes, including the structural genes encoding neurofilament proteins and synaptic vesicle proteins, regulatory genes encoding signaling components beta-arrestin and p38 MAPK, and stress-response genes encoding hsp70. These results show that heme and heme deficiency affect the expression of diverse genes in a cell-type specific manner in mammalian cells, and that heme, although needed at different levels, is critical for both erythropoiesis and neurogenesis. These studies provide insights into how heme may act to control diverse regulatory processes in mammals.

Actins↗
PubMed2004

Hydroxylamine and hydrazine bind directly to the heme iron of the heme-heme oxygenase-1 complex.

We investigated whether or not hydroxylamine (HA) and hydrazine (HZ) interact with heme bound to heme oxygenase-1. Anaerobic addition of either HA or HZ to the ferric heme-enzyme complex produced a low-spin heme species. Titration studies at different pHs revealed that the neutral form of each of HA and HZ selectively binds to the heme with dissociation constants of 9.8 and 1.8 mM, respectively. Electron spin resonance analysis suggested that the nitrogen atom of each amine is coordinated to the ferric heme iron. With a concentrated solution of the heme-enzyme complex, however, another species of HA binding appeared, in which the oxygen atom of HA is coordinated to the iron. This species showed an unusual low-spin signal which is similar to that of the ferric hydroperoxide species in the heme oxygenase reaction.

Animals↗
PubMed1997

The unique heme-heme interactions of the homodimeric Scapharca inaequivalvis hemoglobin as probed in the protein reconstituted with unnatural 2,4 heme derivatives.

In the homodimeric hemoglobin from Scapharca, HbI, functional communication between the two heme groups is based on their direct structural linkage across the subunit interface through the heme propionates. The heme-protein interactions have been altered in deutero- and meso-HbI by substituting the vinyl groups at positions 2 and 4 of protoheme with hydrogen and ethyl groups, respectively. In meso-HbI the introduction of the ethyl groups in the heme pocket induces significant alterations in the conformation of the heme peripheral substituents, including the propionates, and in the structure of bound CO, as revealed by the resonance Raman spectra. The functional counterpart of these structural changes is the loss of cooperativity in carbon monoxide binding and in the rate of oxygen dissociation. Oxygen pulse and flash photolysis experiments indicate that meso-HbI is locked in the liganded conformation. It is postulated that the ethyl groups, which occupy a larger volume than vinyl ones, impair the ligand-linked movement of the heme relative to its pocket and in turn the expression of cooperativity. In deutero-HbI structural alterations have not been monitored. Functionally, cooperativity in the CO binding kinetics is increased as if hydrogen atoms at positions 2 and 4 permitted more marked movements of the heme than in the native protein.

Animals↗
PubMed1977

High-pressure liquid chromatography and field desorption mass spectrometry of heme alpha, heme alpha dimethyl ester and acetyl heme alpha dimethyl ester.

Field desorption mass spectrometry was shown to be a valid technique for determining the molecular weights of hemins and hemin esters, as well as of porphyrins. The observed base peaks of ligand-free protoheme IX, protoheme IX dimethyl ester, and protoporphyrin IX dimethyl ester correspond well to the molecular weights of these compounds and the base peak for hematoporphyrin corresponds to the molecular weight of this porphyrin minus two molecules of water. The technique was employed to confirm the molecular weights of heme alpha, heme alpha dimethyl ester, and acetyl heme alpha dimethyl ester. Heme alpha dimethyl ester was prepared by reaction of heme alpha with trimethyloxonium tetrafluoroborate and purified by high-pressure liquid chromatography. The isolated product was converted to the acetylated derivative by reaction with acetic anhydride and was subsequently purified by high-pressure liquid chromatography. A field desorption spectrum of heme alpha shows a base peak at 582 which is in agreement with previous deductions of the structure of this prosthetic group. Base peaks of the heme alpha ester and its acetylated derivative demonstrate that the two carboxyl groups have been methylated and the single hydroxyl group has been acetylated without further alteration of the molecule.

Chromatography, High Pressure Liquid↗
PubMed1976

The induction of heme oxidation in various tissues by trace metals: evidence for the catabolism of endogenous heme by hepatic heme oxygenase.

Cobalt is a potent inducer of hepatic heme oxygenase and concomitantly decreases microsomal cytochrome P-450 content in liver cells. Studies in which microsomal heme was labelled with 14C-beta-aminolevulinic acid showed that the decline observed in P-450 follows the induction of heme oxygenase indicating that endogenous heme is a substrate for this enzyme. The inducing effect of cobalt on heme oxygenase in liver extended to other organs such as heart, renal cortex and medulla, lungs and intestinal mucosa but not to the spleen and brain. In kidney and heart, cobalt resulted in decreases in microsomal and mitochondrial contents of hemoproteins. In addition ALAS activity of kidney was depressed by the metal. Other metals which chelate with sulfhydryl groups (e.g., Cr, Fe, Ni, Cu, Zn, Cd, Hg, Pb) were also shown to induce heart and renal heme oxygenase activities. Covalent binding of cobalt with mercaptans inactivated the metal with respect to its induction of heme oxygenase and degradation of hemoproteins.

Aminolevulinic Acid↗
PubMed2002

Characterization of a direct oxygen sensor heme protein from Escherichia coli. Effects of the heme redox states and mutations at the heme-binding site on catalysis and structure.

A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its alpha-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min(-1) with cAMP, which was optimal at pH 8.5 in the presence of Mg(2+) and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Potentiometric titration of cytochrome-bo type quinol oxidase of Escherichia coli: evidence for heme-heme and copper-heme interaction.

The cytochrome-bo quinol oxidase of Escherichia coli contains a high-spin b-type heme (cytochrome o), a low-spin b-type heme (cytochrome b) and copper. The EPR signal from cytochrome o is axial high spin and when titrated potentiometrically gives a bell-shaped curve. The low-potential side of this curve (Em7 approx. 160 mV) corresponds to the reduction/oxidation of the cytochrome. The high-potential side (Em7 approx. 350 mV) is proposed to be due to reduction/oxidation of a copper center; in the CuII form tight cytochrome o-copper spin coupling results in a net even spin system and loss of the EPR spectrum. Optical spectra of the alpha-bands of the reduced cytochromes at 77 K show that cytochrome b has its maxima at 564 nm when cytochrome o is oxidized but that this shifts to 561 nm when cytochrome o (max. 555 nm) is reduced. Both a heme-copper (cytochrome o-CuII) and a heme-heme (cytochrome o-cytochrome b) interaction are indicated in this quinol oxidase. These results indicate that cytochrome-bo quinol oxidase has a binuclear heme-copper catalytic site and suggest striking structural similarity to subunit I of the cytochrome aa3 system.

Copper↗
PubMed1993

Heme b (protoheme IX) is a precursor of heme a and heme d in Bacillus subtilis.

Bacillus subtilis can synthesise cytochromes containing a-, b-, c- and d-type heme. The biosynthetic pathways of these heme prosthetic groups were investigated by using strains blocked in uroporphyrinogen III synthesis from porphobilinogen or in heme b (protoheme IX) synthesis from uroporphyrinogen III. The results strongly suggest that heme a and heme d are both synthesised from heme b (protoheme IX). They also indicate that B. subtilis contains a novel ferrochelatase involved in the synthesis of siroheme.

Bacillus subtilis↗
PubMed1992

Mutation of the heme-binding crevice of flavocytochrome b2 from Saccharomyces cerevisiae: altered heme potential and absence of redox cooperativity between heme and FMN centers.

Kinetic and thermodynamic properties of yeast flavocytochrome b2 (EC 1.1.2.3) are modified by the product pyruvate, which binds to the flavosemiquinone (FSQ) form of the prosthetic flavin and decreases the thermodynamic driving force for electron transfer from FSQ to heme. Pyruvate inhibits flavocytochrome b2, but the catalytic competence of pyruvate-ligated FSQ in intramolecular electron transfer to heme is unclear; one kinetic study suggested pyruvate prevented this reaction [Tegoni, M, Janot J.-M., & Labeyrie, F. (1990) Eur. J. Biochem. 190, 329-342], while laser flash photolysis indicated pyruvate was essential [Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555]. To address this problem, wild-type (WT) and mutant (L36I) flavocytochromes b2 have been expressed in Escherichia coli. Both forms incorporated heme and FMN prosthetic groups and were catalytically active. The mutation L36I was a conservative substitution within the heme-binding crevice and was designed to alter the midpoint potential (Em) of the heme to alter the pyruvate-FSQ/heme equilibrium. Potentiometric titrations yielded Em values (pH 7.0, 25 degrees C) of +8 and -28 mV for WT and L36I forms, respectively. The FMN midpoint potentials in the absence of pyruvate (-58 mV, n = 2) were identical within experimental error in WT and L36I species and were also identical (+5 mV, n = 1) in the presence of pyruvate. These results indicated the absence of redox cooperativity between FMN and heme.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2001

Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases. Evidence from 31P NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme.

Two classes of cystathionine beta-synthases have been identified in eukaryotes, the heme-independent enzyme found in yeast and the heme-dependent form found in mammals. Both classes of enzymes catalyze a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to produce cystathionine. The role of the heme in the human enzyme and its location relative to the PLP in the active site are unknown. (31)P NMR spectroscopy revealed that spin-lattice relaxation rates of the phosphorus nucleus in PLP are similar in both the paramagnetic ferric (T(1) = 6.34 +/- 0.01 s) and the diamagnetic ferrous (T(1) = 5.04 +/- 0.06 s) enzyme, suggesting that the two cofactors are not proximal to each other. This is also supported by pulsed EPR studies that do not provide any evidence for strong or weak coupling between the phosphorus nucleus and the ferric iron. However, the (31)P signal in the reduced enzyme moved from 5.4 to 2.2 ppm, and the line width decreased from 73 to 16 Hz, providing the first structural evidence for transmission to the active site of an oxidation state change in the heme pocket. These results are consistent with a regulatory role for the heme as suggested by previous biochemical studies from our laboratory. The (31)P chemical shifts of the resting forms of the yeast and human enzymes are similar, suggesting that despite the difference in their heme content, the microenvironment of the PLP is similar in the two enzymes. The addition of the substrate, serine, resulted in an upfield shift of the phosphorus resonance in both enzymes, signaling formation of reaction intermediates. The resting enzyme spectra were recovered following addition of excess homocysteine, indicating that both enzymes retained catalytic activity during the course of the NMR experiment.

Amino Acid Sequence↗