Simple and rapid method for the determination of coproporphyrinogen oxidase activity

Coproporphyrinogen oxidase, the sixth enzyme in the biosynthetic heme pathway, catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX. A reversed-phase high pressure liquid chromatography method was developed to measure coproporphyrinogen oxidase enzymatic activity in rat liver. With this method, the separation, identification and quantification of coproporphyrin III (oxidized substrate) and protoporphyrin IX (oxidized product) present in the assays could be carried out with no need of derivatization and in less than 15 min. Rat and human liver coproporphyrinogen oxidase basal activities determined using this method were 0.41+/-0.05 nmol of protoporphyrin IX/h per mg of hepatic protein and 0.87+/-0.06 protoporphyrin IX/h per mg of hepatic protein, respectively. Kinetic studies showed that optimum pH for rat CPGox is 7.3, and that its activity is linear in the range of protein concentrations and incubation times assayed. The present paper describes a sensitive, specific and rapid fluorometric high performance liquid chromatography method to measure coproporphyrinogen oxidase, which could be applied to the diagnosis of human coproporphyria, and which is also suitable for the study of lead and other metal poisoning that produce alterations in this enzymatic activity.     

The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae.

The oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells is enzyme-dependent. The enzyme, protoporphyrinogen oxidase, associated with purified mitochondria isolated from Saccharomyces cerevisiae was solubilized by sonic treatment in the presence of detergent and partially purified. The molecular weight of the enzyme was 180,000 plus or minus 18,000. The purified preparation could be stored at -20 degrees in the presence of 20% glycerol for several months without loss of activity. Enzyme activity was destroyed by heating above 40 degrees and by proteolytic digestion and irreversible inactivation occurred outside the pH range of 4.0 to 9.5. The pH optimum of the enzymic reaction was 7.45 and the value of the Michaelis constant was approximately 4.8 muM. Protoporphyrinogen oxidase did not catalyse the oxidation of coproporphyrinogen I or III or uroporphyrinogen I or III and the rate of enzymic oxidation of mesoporphyrinogen IX was less than 20% of that observed with protoporphyrinogen IX. The presence of thiol groups in the enzyme system was indicated but no metal ion or other cofactor requirement was demonstrated. Enzyme activity was insensitive to cyanide, 2,4-dinitrophenol, and azide whereas it was inhibited in the presence of Cu-2+ or Co-2+ ions, high ionic strength, heme, or hemin.     

Porphyrin Accumulation and Export by Isolated Barley (Hordeum vulgare) Plastids (Effect of Diphenyl Ether Herbicides)

We have investigated the formation of porphyrin intermediates by isolated barley (Hordeum vulgare) plastids incubated for 40 min with the porphyrin precursor 5-aminolevulinate and in the presence and absence of a diphenylether herbicide that blocks protoporphyrinogen oxidase, the enzyme in chlorophyll and heme synthesis that oxidizes protoporphyrinogen IX to protoporphyrin IX. In the absence of herbicide, about 50% of the protoporphyrin IX formed was found in the extraplastidic medium, which was separated from intact plastids by centrifugation at the end of the incubation period. In contrast, uroporphyrinogen, an earlier intermediate, and magnesium protoporphyrin IX, a later intermediate, were located mainly within the plastid. When the incubation was carried out in the presence of a herbicide that inhibits protoporphyrinogen oxidase, protoporphyrin IX formation by the plastids was completely abolished, but large amounts of protoporphyrinogen accumulated in the extraplastidic medium. To detect extraplastidic protoporphyrinogen, it was necessary to first oxidize it to protoporphyrin IX with the use of a herbicide-resistant protoporphyrinogen oxidase enzyme present in Escherichia coli membranes. Protoporphyrinogen is not detected by some commonly used methods for porphyrin analysis unless it is first oxidized to protoporphyrin IX. Protoporphyrin IX and protoporphyrinogen found outside the plastid did not arise from plastid lysis, because the percentage of plastid lysis, measured with a stromal marker enzyme, was far less than the percentage of these porphyrins in the extraplastidic fraction. These findings suggest that of the tetrapyrrolic intermediates synthesized by the plastids, protoporphyrinogen and protoporphyrin IX, are the most likely to be exported from the plastid to the cytoplasm. These results help explain the extraplastidic accumulation of protoporphyrin IX in plants treated with photobleaching herbicides. In addition, these findings suggest that plastids may export protoporphyrinogen or protoporphyrin IX for mitochondrial heme synthesis.     

A radiochemical method for the measurement of coproporphyrinogen oxidase and the utilization of substrates other than coproporphyrinogen III by the enzyme from rat liver.

[14C2]Coproporphyrin III, 14C-labelled in the carboxyl carbon atoms of the 2- and 4-propionate substituents, was prepared by stepwise modification of the vinyl groups of protoporphyrin IX. The corresponding porphyrinogen was used as substrate in a specific sensitive assay for coproporphyrinogen oxidase (EC 1.3.3.3) in which the rate of production of 14CO2 is measured. With this method, the Km of the enzyme from rat liver for coproporphyrinogen III is 1.2 micron. Coproporphyrin III is a competitive inhibitor of the enzyme (Ki 7.6 micron). Apparent Km values for other substrates were measured by a mixed-substrate method: that for coproporphyrinogen IV is 0.9 micron and that for harderoporphyrinogen 1.6 micron. Rat liver mitochondria convert pentacarboxylate porphyrinogen III into dehydroisocoproporphyrinogen at a rate similar to that for the formation of protoporphyrinogen IX from coproporphyrinogen III. Mixed-substrate experiments indicate that this reaction is catalysed by coproporphyrinogen oxidase and that the Km for this substrate is 29 micron. It is suggested that the ratio of the concentration of pentacarboxylate porphyrinogen III to coproporphyrinogen III in the hepatocyte determines the relative rates of formation of dehydroisocoproporphyrinogen and protoporphyrinogen IX.     

A high-performance-liquid-chromatographic method for the assay of coproporphyrinogen oxidase activity in rat liver.

An h.p.l.c. method was developed for the assay of coproporphyrinogen oxidase activity in rat liver. The protoporphyrinogen IX formed is completely oxidized to protoporphyrin IX for separation and quantification by reversed-phase chromatography with mesoporphyrin as the internal standard. The Km of coproporphrinogen oxidase is 1.01 +/- 0.23 microM. The activities are 4.07 +/- 0.40 nmol of protoporphyrin IX/h per mg of mitochondrial protein and 224 +/- 19 nmol of protoporphyrin IX/h per g of liver tissue homogenate. The method is sensitive enough for measuring enzyme activity in small amounts of human tissue from needle biopsy.     

Terminal steps of haem biosynthesis

The terminal three steps in haem biosynthesis are the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, followed by the six-electron oxidation of protoporphyrinogen to protoporphyrin IX, and finally the insertion of ferrous iron to form haem. Interestingly, Nature has evolved distinct enzymic machinery to deal with the antepenultimate (coproporphyrinogen oxidase) and penultimate (protoporphyrinogen oxidase) steps for aerobic compared with anaerobic organisms. The terminal step is catalysed by the enzyme ferrochelatase. This enzyme is clearly conserved with regard to a small set of essential catalytic residues, but varies significantly with regard to size, subunit composition, cellular location and the presence or absence of a [2Fe-2S] cluster. Coproporphyrinogen oxidase and protoporphyrinogen oxidase are reviewed with regard to their enzymic and physical characteristics. Ferrochelatase, which is the best characterized of these three enzymes, will be described with particular emphasis paid to what has been learned from the crystal structure of the Bacillus subtilis and human enzymes.     

Oxygen-dependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese

During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.     

Evidence that the coproporphyrinogen oxidase activity of rat liver is situated in the intermembrane space of mitochondria.

Preferential rupture of the outer membrane of mitochondria from rat liver releases coproporphyrinogen oxidase in parallel with components of the intermembrane space. Coproporphyrinogen III enters the mitochondrion through the freely-permeable outer membrane. Either protoporphyrinogen IX or protoporphyrin IX must then cross the inner membrane before haem synthesis can be completed.     

The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria.

Protoporphyrinogen oxidase, an enzyme which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells, has been found in several mammalian tissues. It has been extracted from rat liver mitochondria by sonication in the presence of salt and detergent and partially purified. The enzyme is similar in many respects to yeast protoporphyrinogen oxidase. Based on its behavior on Sephadex G-200 the molecular weight of the enzyme is approximately 35,000. Catalysis by protoporphyrinogen oxidase was specific for proteoporphyrinogen IX (apparent Km of 11 muM) and proceeded maximally at pH 8.6 to 8.7. The effect of temperature on enzyme activity plotted according to Arrhenius gave a value of E of 9,100 calories per mol. Enzyme activity was inhibited in the presence of high salt concentrations and temperatures above 45 degrees. Oxygen was essential for protoporphyrinogen oxidase activity and an alternative elevtron acceptor has not yet been found. No requirement for a metal or other cofactor could be demonstrated. The presence of monothiol groups was indicated; however, it is not known whether the thiol groups are involved directly in the binding of substrate to the enzyme.     

Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides.

Diphenyl ether herbicides induce an accumulation of protoporphyrin IX in plant tissues. By analogy to human porphyria, the accumulation could be attributed to decreased (Mg or Fe)-chelatase or protoporphyrinogen oxidase activities. Possible effects of acifluorfen-methyl on these enzymes were investigated in isolated corn (maize, Zea mays) etioplasts, potato (Solanum tuberosum) and mouse mitochondria, and yeast mitochondrial membranes. Acifluorfen-methyl was strongly inhibitory to protoporphyrinogen oxidase activities whatever their origins [concn. causing 50% inhibition (IC50) = 4 nM for the corn etioplast enzyme]. By contrast, it was roughly 100,000 times less active on (Mg or Fe)-chelatase activities (IC50 = 80-100 microM). Our results lead us to propose protoporphyrinogen oxidase as a cellular target for diphenyl ether herbicides.     

Bacillus subtilis HemY is a peripheral membrane protein essential for protoheme IX synthesis which can oxidize coproporphyrinogen III and protoporphyrinogen IX.

The hemY gene of the Bacillus subtilis hemEHY operon is essential for protoheme IX biosynthesis. Two previously isolated hemY mutations were sequenced. Both mutations are deletions affecting the hemY reading frame, and they cause the accumulation of coproporphyrinogen III or coproporphyrin III in the growth medium and the accumulation of trace amounts of other porphyrinogens or porphyrins intracellularly. HemY was found to be a 53-kDa peripheral membrane-bound protein. In agreement with recent findings by Dailey et al. (J. Biol. Chem. 269:813-815, 1994) B. subtilis HemY protein synthesized in Escherichia coli oxidized coproporphyrinogen III and protoporphyrinogen IX to coproporphyrin and protoporphyrin, respectively. The protein is not a general porphyrinogen oxidase since it did not oxidize uroporphyrinogen III. The apparent specificity constant, kcat/Km, for HemY was found to be about 12-fold higher with coproporphyrinogen III as a substrate compared with protoporphyrinogen IX as a substrate. The protoporphyrinogen IX oxidase activity is consistent with the function of HemY in a late step of protoheme IX biosynthesis, i.e., HemY catalyzes the penultimate step of the pathway. However, the efficient coproporphyrinogen III to coproporphyrin oxidase activity is unexplained in the current view of protoheme IX biosynthesis.     

The mitochondrial location of protoporphyrinogen oxidase

Using the digitonin method and subsequent fractionation of rat liver mitochondria, protoporphyrinogen oxidase (penultimate enzyme in the heme biosynthesis pathway) was found to be closely associated with the mitochondrial inner membrane fraction. Chemical treatment with non-specific probes (trypsin and diazobenzene sulfonate) of either intact or inverted mitoplasts, indicated that protoporphyrinogen oxidase was anchored within the lipid bilayer of the inner membrane. Protoporphyrinogen had an equal access to the active site of the enzyme from both sides of the inner membrane and its transformation to protoporphyrin did not appear to be energy-dependent. Studies of protoporphyrinogen synthesis from exogenously added coproporphyrinogen in either intact or hypoosmotically treated mitochondria underlined the importance of the peculiar submitochondrial location of coproporphyrinogen oxidase and protoporphyrinogen oxidase for the transfer of substrates to the inner membrane.     

High-performance liquid chromatographic assays for protoporphyrinogen oxidase and ferrochelatase in human leucocytes

Rapid, sensitive and specific high-performance liquid chromatographic assays are described for protoporphyrinogen oxidase and ferrochelatase in human leucocytes. The enzyme reaction products were separated and quantitated by reversed-phase high-performance liquid chromatography with fluorescence detection. The optimal pH for the protoporphyrinogen oxidase assay was 8.6 and the Michaelis constant for protoporphyrinogen IX was 9.78 ± 0.96 μM (mean ± S.D.). The mean (± S.D.) activity of protoporphyrinogen oxidase in fourteen apparently healthy subjects was 0.146 ± 0.023 nmol protoporphyrin IX per min per mg protein. In one patient with variegate porphyria, the activity was 0.028 nmol protoporphyrin IX per min per mg protein. The optimal pH for ferrochelatase was 7.4 and with protoporphyrin and Zn²⁺ as substrates, the Michaelis constants were 1.49 and 8.33 μM, respectively. The mean activity of ferrochelatase in ten control subjects was 0.24 nM Zn—protoporphyrin or 2.05 nM Zn—mesoporphyrin formed per h per mg protein.     

A soybean coproporphyrinogen oxidase gene is highly expressed in root nodules

In plants the enzyme coproporphyrinogen oxidase catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s).We have isolated a soybean coproporphyrinogen oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene. The coproporphyrinogen oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa. The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence. The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide.A full-length coproporphyrinogen oxidase cDNA clone complements a yeast mutant deleted of the coproporphyrinogen oxidase gene, thus demonstrating the function of the soybean protein.The soybean coproporphyrinogen oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear. The coproporphyrinogen oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots.The high level of coproporphyrinogen oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation.     

A new assay for protoporphyrinogen oxidase — Evidence for a total deficiency in that activity in a heme-less mutant of Saccharomyces cerevisiae

A new spectrophotometric assay for protoporphyrinogen oxidase activity has been developed, involving enzymatic generation of protoporphyrinogen in the incubation medium. This assay, more sensitive and reliable than those previously described, can be used to measure this activity in yeast mitochondrial membranes, rat liver mitochondria and $\text{E. coli}$ membranes. By measuring protoporphyrinogen oxidase activity in different wild type and heme-mutant yeast strains, it was shown that 1) one heme-mutant was totally lacking this activity, 2) different factors might control its level in yeast.     

Inhibition of plant protoporphyrinogen oxidase by the herbicide acifluorfen-methyl

The effect of acifluorfen-methyl on tetrapyrrole synthesis in greening chloroplasts of Cucumis sativus was examined. Formation of Mg-proto-porphyrin IX from δ-aminolevulinate was reduced 98% by 10 micromolar acifluorfen-methyl. Conversion of protoporphyrin IX to Mg-protoporphyrin IX was unaffected, but protoporphyrin IX synthesis from δ-aminolevulinate was blocked, indicating a site of inhibition prior to the Mg-chelatase. The enzymic oxidation of protoporphyrinogen IX to protoporphyrin IX was highly sensitive to acifluorfen-methyl, indicating that the site of action of the herbicide is the protoporphyrinogen oxidase. (© 1989 FMC Corporation. All rights reserved.)     

Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley

In barley (Hordeum vulgare L.) root cells, activity for oxidizing protoporphyrinogen to protoporphyrin (protoporphyrinogen oxidase), a step in chlorophyll and heme synthesis, was found both in the crude mitochondrial fraction and in a plasma membrane enriched fraction separated by a sucrose gradient technique utilized for preparing plasma membranes. The specific activity (expressed as nanomoles of protoporphyrin formed per hour per milligram protein) in the mitochondrial fraction was 8 and in the plasma membrane enriched fraction was 4 to 6. The plasma membrane enriched fraction exhibited minimal cytochrome oxidase activity and no carotenoid content, indicating little contamination with mitochondrial or plastid membranes. Etioplasts from etiolated barley leaves exhibited a protoporphyrinogen oxidase specific activity of 7 to 12. Protoporphyrinogen oxidase activity in the barley root mitochondrial fraction and etioplast extracts was more than 90% inhibited by assay in the presence of the diphenyl ether herbicide acifluorfen methyl, but the activity in the plasma membrane enriched fraction exhibited much less inhibition by this herbicide (12 to 38% inhibition) under the same assay conditions. Acifluorfen-methyl inhibition of the organellar (mitochondrial or plastid) enzyme was maximal upon preincubation of the enzyme with 4 mm dithiothreitol, although a lesser degree of inhibition was noted if the organellar enzyme was preincubated in the presence of other reductants such as glutathione or ascorbate. Acifluorfen-methyl caused only 20% inhibition if the enzyme was preincubated in buffer without reductants. Incubation of barley etioplast extracts with the earlier tetrapyrrole precursor coproporphyrinogen and acifluorfen-methyl resulted in the accumulation of protoporphyrinogen, which could be converted to protoporphyrin even in the presence of the herbicide by the addition of the plasma membrane enriched fraction from barley roots. These findings have implications for the toxicity of diphenyl ether herbicides, whose light induced tissue damage is apparently caused by accumulation of the photoreactive porphyrin intermediate, protoporphyrin, when the organellar protoporphyrinogen oxidase enzyme is inhibited by herbicides. Our results suggest that the protoporphyrinogen that accumulates as a result of herbicide inhibition of the organellar enzyme can be oxidized to protoporphyrin by a protoporphyrinogen oxidizing activity that is located at sites such as the plasma membrane, which is much less sensitive to inhibition by diphenylether herbicides.