Zinc chelatase in human lymphocytes: detection of the enzymatic defect in erythropoietic protoporphyria

We describe a fluorometric assay for heme synthetase, the enzyme that is genetically deficient in erythropoietic protoporphyria. The method, which can readily detect activity in 1 microliter of packed human lymphocytes, is based on the formation of zinc protoheme from protoporphyrin IX. That zinc chelatase and ferrochelatase activities reside in the same enzyme was shown by the competitive action of ferrous ions and the inhibitory effects of N-methyl protoporphyrin (a specific inhibitor of heme synthetase) on zinc chelatase. The Km for zinc was 11 micrograms and that for protoporphyrin IX was 6 microM. The Ki fro ferrous ions was 14 microM. Zinc chelatase was reduced to 15.3% of the mean control activity in lymphocytes obtained from patients with protoporphyria, thus confirming the defect of heme biosynthesis in this disorder. The assay should prove to be useful for determining heme synthetase in tissues with low specific activity and to investigate further the enzymatic defect in protoporphyria.     

Cloning and characterization of chironomidae ferrochelatase: Copper activation of the purified ferrochelatase

All organisms utilize ferrochelatase (EC 4.99.1.1) to catalyze the insertion of ferrous iron into protoposphyrin IX in the terminal step of the heme biosynthetic pathway. Different metal-binding affinity for the enzyme leads to changes in enzyme activity. In this work, we have cloned and over-expressed the enzyme from chironomidae in E. coli. The enzyme was purified and characterized. The recombinant enzyme showed higher enzymatic activity (four-fold increase) in the presence of copper ions and unaffected by calcium ions. Other divalent metal ions including magnesium, manganese, lead, reduced the enzyme activity by >60%. Over 90% of the enzyme activity was inhibited by Zn2+. The sequence alignment of amino acid residues reveals 83% homology with other ferrochelatases. The results of electron proton resonance (EPR) analysis showed that Fe2+ ion was present in the cluster of the recombinant enzyme complex. The recombinant enzyme also contained the [2Fe-2S] center with two-fold higher enzymatic activity than human ferrochelatase.     

Association of ferrochelatase with Complex I in bovine heart mitochondria

The location of ferrochelatase in bovine heart mitochondria has been studied. When the mitochondria were fractionated into Complexes I, II and III, ferrochelatase activity was only found in Complex I. Complex I also showed heme synthesis from ferric ion in the presence of NADH as an electron donor. Immunoblot experiments confirmed the presence of ferrochelatase in Complex I, but not in Complexes II or III. Some phospholipids, including phosphatidylserine and cardiolipin, stimulated NADH-dependent heme synthesis from ferric ion. When purified ferrochelatase was incubated with the low molecular weight form of NADH dehydrogenase prepared from Complex I, heme synthesis from ferric ion occurred by the addition of NADH. FMN markedly elevated the synthesis. These results indicate that ferrous ion is produced by NADH oxidation in Complex I and is then utilized for heme synthesis by ferrochelatase.     

Reconstitution of heme-synthesizing activity from ferric ion and porphyrins, and the effect of lead on the activity

We examined the activity of heme synthesis when ferrochelatase purified from rat liver mitochondria was incubated with ferric chloride and mesoporphyrin IX as substrates in the absence of reducing reagents. In the presence of the NADH dehydrogenase-rich fraction and NAD(P)H, mesoheme was synthesized; the addition of FMN or FAD markedly enhanced the activity. These results indicate that the NAD(P) H-oxidizing system reduces ferric ion to ferrous ion. This ferrous ion is then utilized for heme synthesis by ferrochelatase. The effect of lead on NAD(P)H-dependent heme synthesis was also examined. Lead reduced NAD(P)H-dependent heme synthesis by 50% at 10(-5) M, but had no effect when ferrous ion was used as substrate. Zn-Porphyrin synthesis was not changed in the presence of Pb2+ at 10(-5) M. Thus, heme synthesis from ferric ion was more susceptible to Pb2+ than heme synthesis from ferrous ion.     

Regulation of the expression of human ferrochelatase by intracellular iron levels.

Mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of a ferrous ion into protoporphyrin and contains a labile [2Fe-2S] cluster center at the C-terminus. To clarify the roles of the iron-sulfur cluster in the expression of mammalian ferrochelatase, enzyme activity in human erythroleukemia K562 cells under iron-depleted conditions was examined. Treatment of cells with an iron chelator, desferrioxamine, resulted in a decrease in enzyme activity, in a dose- and time-dependent manner. Heme content decreased during desferrioxamine treatment of the cells. Addition of ferric ion-nitrilotriacetate [Fe (III)NTA] to desferrioxamine-containing cultures led to restoration of the reduction in the enzyme activity. While RNA blots showed that the amount of ferrochelatase mRNA remained unchanged during these treatments, the amount of ferrochelatase decreased with a concomitant decrease in enzyme activity. When full-length human ferrochelatase was expressed in Cos7 cells, the activity was found mainly in the mitochondria and was decreased markedly by treatment with desferrioxamine. The activity in Cos7 cells expressing human ferrochelatase in cytoplasm decreased with desferrioxamine, but to a lesser extent. When Escherichia coli ferrochelatase, which lacks the iron-sulfur cluster, was expressed in Cos7 cells, the activity did not change following any treatment. Conversely, the addition of Fe (III)NTA to the culture of K562 and Cos7 cells led to an increase in ferrochelatase activity. These results indicate that the expression of mammalian ferrochelatase is regulated by intracellular iron levels, via the iron-sulfur cluster center at the C-terminus, and this contributes to the regulation of the biosynthesis of heme at the terminal step.     

gamma-Butyrobetaine hydroxylase and the protective role of glutathione peroxidase

The selenoenzyme glutathione peroxidase in the presence of GSH effectively replaced catalase in the in vitro assay for gamma-butyrobetaine hydroxylase. Quantitatively, glutathione peroxidase was an order of magnitude more efficient than catalase, with maximal activity at less than 0.1 microM glutathione peroxidase in a standard reaction. Glutathione peroxidase prevented the loss of gamma-butyrobetaine hydroxylase during preliminary incubation with ferrous ions but without other substrates as well as in the course of the reaction. Regardless of whether glutathione peroxidase or catalase was present in the assay, the ascorbate concentrations needed to achieve half-maximal rates were similar (about 1 mM). Phosphate stimulated the rate of L-carnitine synthesis. Ferrous ion saturation indicated a pronounced effect of phosphate on the maximal velocity of the enzyme-catalyzed reaction, but its mechanism of action remains to be elucidated. Based on the subcellular distribution of gamma-butyrobetaine hydroxylase, catalase, and glutathione peroxidase, the role of glutathione peroxidase assumes importance. However, initial studies indicated that the assayable activity of liver gamma-butyrobetaine hydroxylase and L-carnitine concentrations in liver, blood plasma, and muscle were not significantly altered in selenium-deficient rats.     

A continuous anaerobic fluorimetric assay for ferrochelatase by monitoring porphyrin disappearance

A continuous spectrofluorimetric assay for determining ferrochelatase activity has been developed using the physiological substrates ferrous iron and protoporphyrin IX under strictly anaerobic conditions. In contrast to heme, the product of the ferrochelatase-catalyzed reaction, protoporphyrin IX is fluorescent, and therefore the progress of the reaction can be monitored by following the decrease in protoporphyrin fluorescence intensity (with excitation and emission wavelengths at 505 and 635 nm, respectively). This continuous fluorimetric assay detects activities as low as 0.01 nmol porphyrin consumed min(-1), representing an increase in sensitivity of up to two orders of magnitude over the currently used, discontinuous assays. The determination of the steady-state kinetic parameters of ferrochelatase yielded K(m)(PPIX)=1.4+/-0.2 microM, K(m)(Fe(2+))=1.9+/-0.3 microM, and k(cat)=4.0+/-0.3 min(-1). In addition to its applicability for acquisition of kinetic data to characterize ferrochelatase and recombinant variants, this new method should permit detection of low concentrations of ferrochelatase in biological samples.     

Characterization of the hydroperoxide-reducing activity of human plasma

A peroxidase was identified in human plasma using a novel peroxidase assay. In this assay both the substrate 5-phenyl-4-pentenyl hydroperoxide (PPHP) and its reduction product, 5-phenyl-4-pentenyl alcohol (PPA) are quantitated by HPLC. Substrate specificity studies indicated that the peroxidase requires glutathione as reducing substrate. No reduction was detected using the classical heme peroxidase reducing substrates, phenol and hydroquinone. Peroxidase activity was not due to glutathione transferases. Failure to saturate the peroxidase activity with reduced glutathione and inhibition by Cd+2 indicated that it is probably selenium dependent. The enzyme appears to be different from erythrocyte glutathione peroxidase based on kinetic and immunological experiments. The apparent Km values for PPHP are 25 microM for erythrocyte peroxidase and 54 microM for plasma peroxidase at 0.5 mM reduced glutathione. Anti-peroxidase prepared against bovine erythrocyte glutathione peroxidase partially inhibited human erythrocyte peroxidase but did not inhibit human plasma peroxidase.     

Effect of sulfhydryl group modification on the activity of bovine ferrochelatase

The role of sulfhydryl groups in the activity of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), has been examined by using a variety of sulfhydryl group-specific reagents. The enzyme is rapidly inactivated in a pseudo-first order reaction by N-ethylmaleimide and monobromobimane and more slowly by iodoacetamide and bromotrimethylammoniobimane. Reaction with [3H]N-ethylmaleimide indicates that modification of a single sulfhydryl group is sufficient to inactivate bovine ferrochelatase. The enzyme is protected from inactivation by one substrate, ferrous iron, but not by the porphyrin substrate. Mercury and arsenite are reversible inhibitors. The fluorescence of the bound bimane is blue shifted 8 nm from that obtained in aqueous solutions and is sensitive to quenching by iodide.     

Black tea extract supplementation decreases oxidative damage in Jurkat T cells

The purpose of this study was to investigate the protective effect of black tea (BT) extract against induced oxidative damage in Jurkat T-cell line. Cells supplemented with 10 or 25 mg/L BT were subjected to oxidation with ferrous ions. Malondialdehyde (MDA) production as marker of lipid peroxidation, DNA single strand breaks as marker of DNA damage, and modification of the antioxidant enzyme activity, glutathione peroxidase (GPX) were measured. Results show the efficacy of BT polyphenols to decrease DNA oxidative damage and to affect GPX activity (P<0.05), while no effect was shown on MDA production. The succeeding investigation of the activity of caffeine and epigallocatechin gallate demonstrated their antioxidant potential with respect to the cellular markers evaluated. In conclusion, this study supports the protective effect of BT against ferrous ions induced oxidative damage to DNA and the ability of BT to affect the enzyme antioxidant system of Jurkat cells.     

Spectral characterization of manganese peroxidase, an extracellular heme enzyme from the lignin-degrading basidiomycete, Phanerochaete chrysosporium

Manganese peroxidase (MnP) is a component of the lignin degradation system of the basidiomycetous fungus, Phanerochaete chrysosporium. This novel MnII-dependent extracellular enzyme (Mr = 46,000) contains a single protoporphyrin IX prosthetic group and oxidizes phenolic lignin model compounds as well as a variety of other substrates. To elucidate the heme environment of this enzyme, we have studied its electron paramagnetic resonance and resonance Raman spectroscopic properties. These studies indicate that the native enzyme is predominantly in the high-spin ferric form and has a histidine as fifth ligand. The reduced enzyme has a high-spin, pentacoordinate ferrous heme. Fluoride and cyanide readily bind to the sixth coordination position of the heme iron in the native form, thereby changing MnP into a typical high-spin, hexacoordinate fluoro adduct or a low-spin, hexacoordinate cyano adduct, respectively. EPR spectra of 14NO- and 15NO-adducts of ferrous MnP were compared with those of horseradish peroxidase (HRP); the presence of a proximal histidine ligand was confirmed from the pattern of superhyperfine splittings of the NO signals centered at g approximately equal to 2.005. The appearance of the FeII-His stretch at approximately 240 cm-1 and its apparent lack of deuterium sensitivity suggest that the N delta proton of the proximal histidine of the enzyme is more strongly hydrogen bonded than that of oxygen carrier globins and that this imidazole ligand may be described as having a comparatively strong anionic character. Although resonance Raman frequencies for the spin- and coordination-state marker bands of native MnP, nu 3 (1487), nu 19 (1565), and nu 10 (1622 cm-1), do not fall into frequency regions expected for typical penta- or hexacoordinate high-spin ferric heme complexes, ligation of fluoride produces frequency shifts of these bands very similar to those observed for cytochrome c peroxidase and HRP. Hence, these data strongly suggest that the iron in native MnP is predominantly high-spin pentacoordinate. Analysis of the Raman frequencies indicates that the dx2-y2 orbital of the native enzyme is at higher energy than that of metmyoglobin. These features of the heme in MnP must be favorable for the peroxidase catalytic mechanism involving oxidation of the heme iron to FeIV. Consequently, it is most likely that the heme environment of MnP resembles those of HRP, cytochrome c peroxidase, and lignin peroxidase.     

Subcellular localization of two types of ferrochelatase in cucumber

It is widely believed that ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous ion into protoporphyrin IX to form protoheme, exists in both plastids and mitochondria of higher plants. By in vitro import assay with isolated pea (Pisum sativum L.) organelles, it has been proposed that one of two isoforms of ferrochelatase (type 1) is dual-targeted into both plastids and mitochondria, and functions for heme biosynthesis in the both organelles. Recently, however, mitochondrial targeting of ferrochelatase is being disputed since pea mitochondria appeared to accept a variety of chloroplast proteins including the type-1 ferrochelatase of Arabidopsis thaliana (L.) Heynh. To clarify the precise subcellular localization of ferrochelatase in higher plants, here we investigated the subcellular localization of two types of ferrochelatase (CsFeC1 and CsFeC2) in cucumber (Cucumis sativus L.). In cotyledons, a significant level of specific ferrochelatase activity was detected in thylakoid membranes, but only a trace level of activity was detectable in mitochondria. Western blot analysis with specific antibodies showed that anti-CsFeC2 antiserum cross-reacted with plastids in photosynthetic and non-photosynthetic tissues. Anti-CsFeC1 did not cross-react with mitochondria, but CsFeC1 was clearly detectable in plastids from non-photosynthetic tissues. In situ transient-expression assays using green fluorescent protein demonstrated that, as well as CsFeC2, the N-terminal transit peptide of CsFeC1 targeted the fusion protein solely into plastids, but not into mitochondria. These results demonstrated that in cucumber both CsFeC1 and CsFeC2 are solely targeted into plastids, but not into mitochondria. Screening of a cucumber genomic or cDNA library did not allow any other ferrochelatase homologous gene to be isolated. The data presented here imply the reconsideration of mitochondrial heme biosynthesis in higher plants.     

Automated continuous-flow colorimetric determination of glutathione peroxidase with dichloroindophenol

Automation of the glutathione peroxidase enzyme assay has been problematical. Although such methods have been reported, they do not give equivalent results to the standard manual assay, wherein glutathione oxidation is coupled to NADPH oxidation via glutathione reductase. We report here the development of a fully automated, continuous-flow, colorimetric method for glutathione peroxidase assays in which glutathione oxidation is monitored by its effect on the reaction of glutathione with the colorimetric reagent 2,6-dichloroindophenol. This method has a linear response to glutathione peroxidase over an 800-fold range of enzyme concentrations. Results of assays done by this method in erythrocyte and plasma samples correlate well with the standard manual coupled assay (r = 0.997 and 0.923, respectively), with no evidence of systematic errors. The assay works equally well with hydrogen peroxide or cumene hydroperoxide as substrate and shows the same selectivity toward glutathione S-transferases as the standard coupled assay. The within-day repeatability and the between-day reproducibility were estimated as 1.1 to 6.4% and 1.3 to 7.1% (relative standard deviation), respectively. This method is suitable for enzyme determinations in whole blood, erythrocytes, plasma, and serum from rats, rabbits, monkeys, and humans.     

Heme synthase (ferrochelatase) catalyzes the removal of iron from heme and demetalation of metalloporphyrins

The red pigments in meat products, including cooked cured ham, arise from the reaction of myoglobin with nitric oxide generated from exogenous nitrite. Since carcinogenic nitrosoamines may be generated by the treatment of meats with nitrite, the production of nitrite-free meat products is an attractive alternative. Raw dry-cured (Parma) hams are produced by the treatment of meats with salts other than nitrite. Analysis of pigments in raw dry-cured hams reveals that the main pigment is zinc protoporphyrin, suggesting that the conversion of heme to zinc protoporphyrin occurs via an iron-removal reaction from myoglobin heme during the processing of raw hams. Purification of the iron-removal enzyme showed that it was identical to ferrochelatase. Recombinant ferrochelatase in combination with NADH-cytochrome b5 reductase catalyzed NADH-dependent iron-removal reaction from hemin and hemoproteins. Metal ions such as zinc and cobalt were also removed from the corresponding metalloporphyrins. The addition of zinc ions led to the formation of zinc protoporphyrin. In cultured cells, the conversion of zinc mesoporphyrin to mesoheme was observed to be dependent on ferrochelatase and could be markedly induced during erythroid differentiation. This is the first demonstration of a new enzyme reaction, the reverse reaction of ferrochelatase, which may contribute to a new route of the recycling of protoporphyrin and heme in cells.     

Binding of protoporphyrin IX and metal derivatives to the active site of wild-type mouse ferrochelatase at low porphyrin-to-protein ratios

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe(2+) chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.     

Differential response of testicular and ovarian heme oxygenase activity to metal ions

The response of the microsomal heme oxygenase in the testis to metal ions distinctly differed from that of the ovarian source. The activity of the ovarian enzyme in rats treated with Co2+ (250 mumol/kg, 24 h) responded in consonance with that of the liver and the kidney, i.e., heme oxygenase activity was elevated. In contrast, similar treatments did not increase the activity of testicular heme oxygenase. In addition, other metal ions, such as Cu2+, Sn2+, Pb2+, and Hg2+, known for their potency to increase heme oxygenase activity, were ineffective in increasing the enzyme activity in the testis. The unprecedented response of heme oxygenase in the testis to metal ions did not reflect an unusual nature of the enzyme protein insofar as it displayed a similar cofactor requirement and inhibition by known inhibitors of the enzyme activity, such as KCN and NaN3. Moreover, the apparent Km's for oxidation of hematoheme by the testicular and ovarian microsomal fractions were comparable and measured 2.3 and 1.4 microM, respectively. In the testis of Co2+-treated rats, the concentration of cytochrome P-450 in the rough and smooth endoplasmic reticular fractions was significantly decreased. The decrease in the hemoprotein level, however, did not reciprocate the activity of heme oxygenase in the fractions. The inability of metal ions to induce heme oxygenase activity in the testis did not represent the general refractory nature of the enzymes of heme metabolism to metal ions in this organ, since in rats treated with Co2+ the activity of delta-aminolevulinate synthetase was significantly decreased 24 h after treatment. However, the activities of uroporphyrinogen-I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the content of porphyrins were not altered in the testis of rats treated with Co2+. The response of delta-aminolevulinate synthetase in the ovarian tissue to Co2+ treatment contrasted that of the testis. In the ovary, the enzyme activity significantly decreased 6 h after treatment. This decrease was followed by a rebound increase at 24 h after administration of Co2+. The presently described inability of metal ions to induce testicular heme oxygenase activity suggests that the activity of the enzyme in the testis is controlled by factor(s) which differ from those regulating the enzyme activity in other organs, including another steroidogenic organ, the ovary.     

Structure and function of ferrochelatase

Ferrochelatase is the terminal enzyme of the heme biosynthetic pathway in all cells. It catalyzes the insertion of ferrous iron into protoporphyrin IX, yielding heme. In eukaryotic cells, ferrochelatase is a mitochondrial inner membrane-associated protein with the active site facing the matrix. Decreased values of ferrochelatase activity in all tissues are a characteristic of patients with protoporphyria. Point-mutations in the ferrochelatase gene have been recently found to be associated with certain cases of erythropoietic protoporphyria. During the past four years, there have been considerable advances in different aspects related to structure and function of ferrochelatase. Genomic and cDNA clones for bacteria, yeast, barley, mouse, and human ferrochelatase have been isolated and sequenced. Functional expression of yeast ferrochelatase in yeast strains deficient in this enzyme, and expression inEscherichia coli and in baculovirusinfected insect cells of different ferrochelatase cDNAs have been accomplished. A recently identified (2Fe-2S) cluster appears to be a structural feature shared among mammalian ferrochelatases. Finally, functional studies of ferrochelatase site-directed mutants, in which key amino acids were replaced with residues identified in some cases of protoporphyria, will be summarized in the context of protein structure.     

Regulation of aldoxime dehydratase activity by redox-dependent change in the coordination structure of the aldoxime-heme complex

Phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1 (OxdB) catalyzes the dehydration of Z-phenylacetaldoxime (PAOx) to produce phenylacetonitrile. OxdB contains a protoheme that works as the active center of the dehydration reaction. The enzymatic activity of ferrous OxdB was 1150-fold higher than that of ferric OxdB, indicating that the ferrous heme was the active state in OxdB catalysis. Although ferric OxdB was inactive, the substrate was bound to the ferric heme iron. Electron paramagnetic resonance spectroscopy revealed that the oxygen atom of PAOx was bound to the ferric heme, whereas PAOx was bound to the ferrous heme in OxdB via the nitrogen atom of PAOx. These results show a novel mechanism by which the activity of a heme enzyme is regulated; that is, the oxidation state of the heme controls the coordination structure of a substrate-heme complex, which regulates enzymatic activity. Rapid scanning spectroscopy using stopped-flow apparatus revealed that a reaction intermediate (the PAOx-ferrous OxdB complex) showed Soret, alpha, and beta bands at 415, 555, and 524 nM, respectively. The formation of this intermediate complex was very fast, finishing within the dead time of the stopped-flow mixer (approximately 3 ms). Site-directed mutagenesis revealed that His-306 was the catalytic residue responsible for assisting the elimination of the hydrogen atom of PAOx. The pH dependence of OxdB activity suggested that another amino acid residue that assists the elimination of the OH group of PAOx would work as a catalytic residue along with His-306.     

Heme-protein covalent bonds in peroxidases and resistance to heme modification during halide oxidation

Plant peroxidases, as typified by horseradish peroxidase (HRP), primarily catalyze the one-electron oxidation of phenols and other low oxidation potential substrates. In contrast, the mammalian homologues such as lactoperoxidase (LPO) and myeloperoxidase primarily oxidize halides and pseudohalides to the corresponding hypohalides (e.g., Br(-) to HOBr, Cl(-) to HOCl). A further feature that distinguishes the mammalian from the plant and fungal enzymes is the presence of two or more covalent bonds between the heme and the protein only in the mammalian enzymes. The functional roles of these covalent links in mammalian peroxidases remain uncertain. We have previously reported that HRP can oxidize chloride and bromide ions, but during oxidation of these ions undergoes autocatalytic modification of its heme vinyl groups that virtually inactivates the enzyme. We report here that autocatalytic heme modification during halide oxidation is not unique to HRP but is a general feature of the oxidation of halide ions by fungal and plant peroxidases, as illustrated by studies with Arthromyces ramosus and soybean peroxidases. In contrast, LPO, a prototypical mammalian peroxidase, is protected from heme modification and its heme remains intact during the oxidation of halide ions. These results support the hypothesis that the covalent heme-protein links in the mammalian peroxidases protect the heme from modification during the oxidation of halide ions.     

Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.