Human Coproporphyrinogen Oxidase Is Not a Metalloprotein

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.

Coproporphyrinogen oxidase (EC 1.3.3.3) catalyzes the sixth step in the heme biosynthetic pathway, the oxidation of coproporphyrinogen III to protoporphyrinogen IX. The activity of this enzyme is deficient in the disease hereditary coproporphyria. The sequence of the cDNA and predicted amino acid sequence of the human coproporphyrinogen oxidase are presented. The human protein sequence contains a region completely homologous to that we obtained by sequencing an 11-amino acid peptide fragment from purified murine liver coproporphyrinogen oxidase. Results of Southern blotting were consistent with the presence of a single human coproporphyrinogen oxidase gene, and Northern blotting demonstrated one transcript of similar size in erythroid and nonerythroid cell lines. Expression of the cDNA coding for the putative mature human coproporphyrinogen oxidase in Escherichia coli resulted in a 17-fold increase in coproporphyrinogen activity over endogenous activity.

Molecular cloning, sequencing and expression of cDNA encoding human coproporphyrinogen oxidase

Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase

A cascade analysis of the interaction of mercury and coproporphyrinogen oxidase (CPOX) polymorphism on the heme biosynthetic pathway and porphyrin production

Porphyrias are inherited or acquired disorders of heme biosynthesis characterized by heme deficiency and accumulation of toxic intermediates. In δ-aminolevulinic acid dehydratase deficiency porphyria (ALADP), patients consistently present elevated urinary δ-aminolevulinic acid (δ-ALA) and coproporphyrin III (COPRO III), yet the mechanistic basis of COPRO III accumulation remains unclear. This metabolic disturbance is also observed in the porphyria-like associated crises in hereditary tyrosinemia type I (HT1). Here, we investigated the effects of δ-ALA, COPRO III, and lead (Pb2+) on human coproporphyrinogen oxidase (CPOX), a key mitochondrial enzyme in heme biosynthesis. Using purified recombinant CPOX, we show that COPRO III binds with high affinity (KD ≈ 2.1 μM) and acts as a competitive inhibitor, while δ-ALA inhibits CPOX at millimolar concentrations through a non-competitive, likely covalent, mechanism. In vivo, δ-ALA accumulation in an HT1 mouse model led to hepatic COPRO III buildup, consistent with our in vitro findings and supporting a synergistic inhibition model in which δ-ALA promotes secondary COPRO III accumulation that further impairs CPOX. Additionally, Pb2+ was found to inactivate CPOX, likely through oxidative damage, providing a molecular explanation for enzyme dysfunction in porphyrin abnormalities in response to lead intoxication. Together, these results identify multiple metabolite- and toxin-dependent mechanisms that converge on CPOX inhibition, offering new insights into the pathophysiology of ALADP, among other porphyrias, lead intoxication, and HT1.

Coproporphyrinogen oxidase (CPOX), the sixth enzyme in the heme-biosynthetic pathway, catalyzes oxidative decarboxylation of coproporphyrinogen to protoporphyrinogen and is located in the intermembrane space of mitochondria. To clarify the importance of CPOX in the regulation of heme biosynthesis in erythroid cells, we established human erythroleukemia K562 cells stably expressing mouse CPOX. The CPOX cDNA-transfected cells had sevenfold higher CPOX activity than cells transfected with vector only. Expression of ferrochelatase and heme content in the transfected cells increased slightly compared with the control. When K562 cells overexpressing CPOX were treated with delta-aminolevulinic acid (ALA), most became benzidine-positive without induction of the expression of CPOX or ferrochelatase, and the heme content was about twofold higher than that in ALA-treated control cells. Increases in cellular heme concomitant with a marked induction of the expression of heme-biosynthetic enzymes, including CPOX, ferrochelatase and erythroid-specific delta-aminolevulinic acid synthase, as well as of alpha-globin synthesis, were observed when cells were treated with transforming growth factor (TGF)beta 1. These increases in the transfected cells were twice those in control cells, indicating that overexpression of CPOX enhanced induction of the differentiation of K562 cells mediated by TGF beta 1 or ALA. Conversely, the transfection of antisense oligonucleotide to human CPOX mRNA into untreated and TGF beta 1-treated K562 cells led to a decrease in heme production compared with sense oligonucleotide-transfected cells. These results suggest that CPOX plays an important role in the regulation of heme biosynthesis during erythroid differentiation.

To maintain photosynthetic competence under copper-deficient conditions, the green alga Chlamydomonas reinhardtii substitutes a heme protein (cytochrome c6) for an otherwise essential copper protein, viz. plastocyanin. Here, we report that the gene encoding coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway, is coordinately expressed with cytochrome c6 in response to changes in copper availability. We have purified coproporphyrinogen oxidase from copper-deficient C.reinhardtii cells, and have cloned a cDNA fragment which encodes it. Northern hybridization analysis confirmed that the protein is nuclear-encoded and that, like cytochrome c6, its expression is regulated by copper at the level of mRNA accumulation. The copper-responsive expression of coproporphyrinogen oxidase parallels cytochrome c6 expression exactly. Specifically, the copper-sensing range and metal selectivity of the regulatory components, as well as the time course of the responses, are identical. Hence, we propose that the expression of these two proteins is controlled by the same metalloregulatory mechanism. Our findings represent a novel metalloregulatory response in which the synthesis of one redox cofactor (heme) is controlled by the availability of another (Cu).

Coproporphyrinogen oxidase (CPOX) catalyzes the two-step decarboxylation of coproporphyrinogen-III to protoporphyrinogen-IX in the heme biosynthetic pathway. Previously we described a specific polymorphism (A814C) in exon 4 of the human CPOX gene (CPOX4) and demonstrated that CPOX4 is associated with both modified urinary porphyrin excretion and increased neurobehavioral deficits among human subjects with low-level mercury (Hg) exposure. Here, we sought to characterize the gene products of CPOX and CPOX4 with respect to biochemical and kinetic properties. Coproporphyrinogen-III was incubated with recombinantly expressed and purified human CPOX and CPOX4 enzymes at various substrate concentrations, with or without Hg(2+) present. Both CPOX and CPOX4 formed protoporphyrinogen-IX from coproporphyrinogen-III; however, the affinity of CPOX4 was twofold lower than that of CPOX (CPOX K(m) = 0.30 microM, V(max) = 0.52 pmol protoporphyrin-IX; CPOX4 K(m) = 0.54 microM, V(max) = 0.33 pmol protoporphyrin-IX). Hg(2+) specifically inhibited the second step of coproporphyrinogen-III decarboxylation (harderoporphyrinogen to protoporphyrinogen-IX) in a dose dependent manner. We also compared the catalytic activities of CPOX and CPOX4 in human liver samples. The specific activities of CPOX in mutant livers were significantly lower (40-50%) than those of either wild-type or heterozygous. Additionally, enzymes from mutant, heterozygous and wild-type livers were comparably inhibited by Hg(2+) (10 microM), decreasing CPOX4 activity to 25% that of the wild-type enzyme. These findings suggest that CPOX4 may predispose to impaired heme biosynthesis, which is limited further by Hg exposure. These effects may underlie increased susceptibility to neurological deficits previously observed in Hg-exposed humans with CPOX4.

Function and structure of rat hepatic coproporphyrinogen oxidase

The biosynthesis of heme is a complex multi-step pathway requiring the efforts of eight enzymes. The initial enzymes in the heme biosynthetic pathway have been well characterized in relation to their mechanisms. Coproporphyrinogen oxidase (Copro'gen oxidase) is one of the last three enzymes in the pathway and is one of the least well understood. Copro'gen oxidase converts Coproporphyrinogen III to protoporphyrinogen IX via oxidative decarboxylation of the 3- and 8- propionic side chain moieties. To further our understanding of the recognition and binding of substrate, Copro'gen oxidase was partially purified from chicken red blood cell hemolysates then incubated with covalent modifiers of specific amino acids. Incubation with tetranitromethane, p-hydroxyphenylglyoxal, N-acetylimidazole, or trinitrobenzenesulfonic acid resulted in substantial reduction of Copro'gen oxidase activity implying the presence of critical tyrosine, arginine and lysine residues. We conclude that these amino acids play important roles in the enzymic mechanism (for both binding and catalysis) of Copro'gen oxidase.

Several proteins are able to bind porphyrins, but because of their function few are interacting specifically. These few proteins are enzymes in the heme biosynthetic pathway and proteins like the so-called Z-protein in the liver. The first enzyme in that pathway that binds a porphyrin-like molecule is uroporphyrinogen synthetase. This enzyme and the enzymes catalyzing the next steps, uroporphyrinogen cosynthetase and uroporphyrinogen decarboxylase are located in the cytoplasm. The last enzymes, coproporphyrinogen oxidase, protoporphyrinogen oxidase and ferrochelatase are located in the mitochondria. Only these last two enzymes are proteins that bind porphyrins. Little is known of the porphyrin binding properties of these enzymes. The Michaelis Menten constants for various porphyrin(ogen)s are summarized in table I.

Comparison of two assay methods for activities of uroporphyrinogen decarboxylase and coproporphyrinogen oxidase

Photodynamic therapy (PDT) with aminolevulinate (ALA) is widely accepted as an effective treatment for superficial carcinomas and pre-cancers. However, PDT is still suboptimal for deeper tumors, mainly due to inadequate ALA penetration and subsequent conversion to PpIX. We are interested in improving the effectiveness of photodynamic therapy (PDT) for deep tumors, using a combination approach (cPDT) in which target protoporphyrin (PpIX) levels are significantly enhanced by differentiation caused by giving Vitamin D or methotrexate (MTX) for 3 days prior to ALAPDT. In LNCaP and MEL cells, a strong correlation between inducible differentiation and expression of C/EBP transcription factors, as well as between differentiation and mRNA levels of CPO (a key heme-synthetic enzyme), indicates the possibility of CPO transcriptional regulation by the C/EBPs. Sequence analysis of the first 1300 base pairs of the murine CPO upstream region revealed 15 consensus C/EBP binding sites. Electrophoretic Mobility Shift Assays (EMSA) proved that these sites form specific complexes that have strong, moderate or weak affinities for C/EBPs. However, in the context of the full-length CPO promoter, inactivation of any type of site (strong or weak) reduced CPO promoter activity (luciferase assay) to nearly the same extent, suggesting cooperative interactions. A comparative analysis of murine and human CPO promoters revealed possible protein-protein interactions between C/EBPs and several neighboring transcription factors such as NFkB, Sp1, AP-1, CBP/p300 and CREB (an enhanceosome complex). Overall, these results confirm that C/EBP’s are important for CPO expression via complex mechanisms which upregulate PpIX and enhance the outcome of cPDT.

The effects of covalent cross-linkers on the enzyme, coproporphyrinogen oxidase, had been previously studied but their role in protecting the enzyme from protease cleavage has not been evaluated. Therefore, we examined how the cross-linker bis (sulfosuccinimidyl) suberate (BS3) affects the ability of trypsin to digest purified, wild type recombinant human coproporphyrinogen oxidase and selected mutants. Following incubation, the apparent molecular weights of peptides were evaluated by SDS-PAGE and enzymatic activity was assessed by spectroscopy following HPLC. For both wild type and mutants, the results indicated that the cross-linker was indeed able to protect against trypsin digestion relative to the enzyme incubated with trypsin in the absence of the cross-linker. These data have implications for the episodic nature of porphyria.