Tyrosyl Radical-Mediated Sequential Oxidative Decarboxylation of Coproporphyrinogen III through PCET: Theoretical Insights into the Mechanism of Coproheme Decarboxylase ChdC.

The peroxide-dependent coproheme decarboxylase ChdC from Geobacillus stearothermophilus catalyzes two key steps in the synthesis of heme b, i.e., two sequential oxidative decarboxylations of coproporphyrinogen III (coproheme III) at propionate groups P2 and P4. In the binding site of coproheme III, P2 and P4 are anchored by different residues (Tyr144, Arg217, and Ser222 for P2 and Tyr113, Lys148, and Trp156 for P4); however, strong experimental evidence supports that the generated Tyr144 radical acts as an unique intermediary for hydrogen atom transfer (HAT) from both reactive propionates. So far, the reaction details are still unclear. Herein, we carried out quantum mechanics/molecular mechanics calculations to explore the decarboxylation mechanism of coproheme III. In our calculations, the coproheme Cpd I, Fe(IV) = O coupled to a porphyrin radical cation (por•+) with four propionate groups, was used as a reactant model. Our calculations reveal that Tyr144 is directly involved in the decarboxylation of propionate group P2. First, the proton-coupled electron transfer (PCET) occurs from Tyr144 to P2, generating a Tyr144 radical, which then abstracts a hydrogen atom from the Cß of P2. The ß-H extraction was calculated to be the rate-limiting step of decarboxylation. It is the porphyrin radical cation (por•+) that makes the PCET from Tyr144 to P2 to be quite easy to initiate the decarboxylation. Finally, the electron transfers from the Cß• through the porphyrin to the iron center, leading to the decarboxylation of P2. Importantly, the decarboxylation of P4 mediated by Lys148 was calculated to be very difficult, which suggests that after the P2 decarboxylation, the generated harderoheme III intermediate should rebind or rotate in the active site so that the propionate P4 occupies the binding site of P2, and Tyr144 again mediates the decarboxylation of P4. Thus, our calculations support the fact that Tyr144 is responsible for the decarboxylation of both P2 and P4.

In order for the light to shine so brightly, the darkness must be present-why do cancers fluoresce with 5-aminolaevulinic acid?

Photodynamic diagnosis and therapy have emerged as a promising tool in oncology. Using the visible fluorescence from photosensitisers excited by light, clinicians can both identify and treat tumour cells in situ. Protoporphyrin IX, produced in the penultimate step of the haem synthesis pathway, is a naturally occurring photosensitiser that visibly fluoresces when exposed to light. This fluorescence is enhanced considerably by the exogenous administration of the substrate 5-aminolaevulinic acid (5-ALA). Significantly, 5-ALA-induced protoporphyrin IX accumulates preferentially in cancer cells, and this enhanced fluorescence has been harnessed for the detection and photodynamic treatment of brain, skin and bladder tumours. However, surprisingly little is known about the mechanistic basis for this phenomenon. This review focuses on alterations in the haem pathway in cancer and considers the unique features of the cancer environment, such as altered glucose metabolism, oncogenic mutations and hypoxia, and their potential effects on the protoporphyrin IX phenomenon. A better understanding of why cancer cells fluoresce with 5-ALA would improve its use in cancer diagnostics and therapies.

Revisiting the Mechanism of the Anaerobic Coproporphyrinogen†III Oxidase HemN.

HemN is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, an intermediate in heme biosynthesis. HemN binds two SAM molecules in the active site, but how these two SAMs are utilized for the sequential decarboxylation of the two propionate groups of coproporphyrinogen III remains largely elusive. Provided here is evidence showing that in HemN catalysis a SAM serves as a hydrogen relay which mediates a radical-based hydrogen transfer from the propionate to the 5'-deoxyadenosyl (dAdo) radical generated from another SAM in the active site. Also observed was an unexpected shunt product resulting from trapping of the SAM-based methylene radical by the vinyl moiety of the mono-decarboxylated intermediate, harderoporphyrinogen. These results suggest a major revision of the HemN mechanism and reveal a new paradigm of the radical-mediated hydrogen transfer in radical SAM enzymology.

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase.

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large-scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe2+ or Mg2+ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen-regulated pufQBALMX operon encoding the reaction centre-light-harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG-immunoprecipitation experiments retrieve a ferrochelatase-PufQ-carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen-limiting conditions. The co-location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light-harvesting polypeptides.

Gametophyte Development Needs Mitochondrial Coproporphyrinogen III Oxidase Function.

Tetrapyrrole biosynthesis is one of the most essential metabolic pathways in almost all organisms. Coproporphyrinogen III oxidase (CPO) catalyzes the conversion of coproporphyrinogen III into protoporphyrinogen IX in this pathway. Here, we report that mutation in the Arabidopsis (Arabidopsis thaliana) CPO-coding gene At5g63290 (AtHEMN1) adversely affects silique length, ovule number, and seed set. Athemn1 mutant alleles were transmitted via both male and female gametes, but homozygous mutants were never recovered. Plants carrying Athemn1 mutant alleles showed defects in gametophyte development, including nonviable pollen and embryo sacs with unfused polar nuclei. Improper differentiation of the central cell led to defects in endosperm development. Consequently, embryo development was arrested at the globular stage. The mutant phenotype was completely rescued by transgenic expression of AtHEMN1 Promoter and transcript analyses indicated that AtHEMN1 is expressed mainly in floral tissues and developing seeds. AtHEMN1-green fluorescent protein fusion protein was found targeted to mitochondria. Loss of AtHEMN1 function increased coproporphyrinogen III level and reduced protoporphyrinogen IX level, suggesting the impairment of tetrapyrrole biosynthesis. Blockage of tetrapyrrole biosynthesis in the AtHEMN1 mutant led to increased reactive oxygen species (ROS) accumulation in anthers and embryo sacs, as evidenced by nitroblue tetrazolium staining. Our results suggest that the accumulated ROS disrupts mitochondrial function by altering their membrane polarity in floral tissues. This study highlights the role of mitochondrial ROS homeostasis in gametophyte and seed development and sheds new light on tetrapyrrole/heme biosynthesis in plant mitochondria.

Oxygen-dependent copper toxicity: targets in the chlorophyll biosynthesis pathway identified in the copper efflux ATPase CopA deficient mutant.

Characterization of a copA(-) mutant in the purple photosynthetic bacterium Rubrivivax gelatinosus under low oxygen or anaerobic conditions, as well as in the human pathogen Neisseria gonorrhoeae identified HemN as a copper toxicity target enzyme in the porphyrin synthesis pathway. Heme synthesis is, however, unaffected by copper under high oxygen tension because of the aerobic coproporphyrinogen III oxidase HemF. Nevertheless, in the copA(-) mutant under aerobiosis, we show that the chlorophyll biosynthesis pathway is affected by excess copper resulting in a substantial decrease of the photosystem. Analyses of pigments and enzyme activity showed that under low copper concentrations, the mutant accumulated protochlorophyllide, suggesting that the protochlorophyllide reductase activity is affected by excess copper. Increase of copper concentration led to a complete lack of chlorophyll synthesis as a result of the loss of Mg-chelatase activity. Both enzymes are widely distributed from bacteria to plants; both are [4Fe-4S] proteins and oxygen sensitive; our data demonstrate their in vivo susceptibility to copper in the presence of oxygen. Additionally, our study provides the understanding of molecular mechanisms that may contribute to chlorosis in plants when exposed to metals. The role of copper efflux systems and the impact of copper on heme and chlorophyll biosynthesis in phototrophs are addressed.

Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that T. gondii CPDH (TgCPDH) potentially functions as an alternative enzyme to perform the same reaction as CPOX under low-oxygen conditions. In this study, we demonstrated that TgCPDH exhibits CPDH activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wild-type and heme-deficient parasites did not alter their intracellular growth under both ambient and low-oxygen conditions. This research marks the first report of a CPDH-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings revealed that it does not directly affect acute Toxoplasma infections in vitro and in vivo. IMPORTANCE: Toxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its life cycle, these parasites must adapt to varying environmental conditions, including situations with low-oxygen levels, such as intestine and spleen tissues. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase (CPDH), suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, CPDH has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying CPDH activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

Identification of a coproporphyrinogen-III oxidase gene and its correlation with nacre color in Hyriopsis cumingii.

Pearl color is an important factor influencing pearl value, and is affected by the nacre color of the shell in Hyriopsis cumingii. Coproporphyrinogen-III oxidase (CPOX) is a key enzyme in porphyrin synthesis, and porphyrins are involved in color formation in different organisms, including in the nacre color of mussels. In this study, a CPOX gene (HcCPOX) was identified from H. cumingii, and its amino acid sequence was found to contain a coprogen-oxidase domain. HcCPOX mRNA was expressed widely in the tissues of white and purple mussels, and the highest expression was found in the gill, followed by the fringe mantle. The expression of HcCPOX in all tissues of purple mussels (except in the middle mantle) was higher than that of white mussels. Strong hybridization signals for HcCPOX were observed in the dorsal epithelial cells of the outer fold of the mantle. The activity of CPOX in the gill, fringe mantle, and foot of purple mussels was significantly higher than that in white mussels. Moreover, the expression of HcCPOX and CPOX activity were decreased in RNA interference experiments. The findings indicate that HcCPOX might contributes to nacre color formation in H. cumingii by being involved in porphyrin synthesis.

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.

Analogues of coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H(2)SO(4)-TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9. Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from coproporphyrinogen oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.

The oxygen-independent coproporphyrinogen III oxidase HemN utilizes harderoporphyrinogen as a reaction intermediate during conversion of coproporphyrinogen III to protoporphyrinogen IX.

During heme biosynthesis the oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of the two propionate side chains on rings A and B of coproporphyrinogen III to the corresponding vinyl groups to yield protoporphyrinogen IX. Here, the sequence of the two decarboxylation steps during HemN catalysis was investigated. A reaction intermediate of HemN activity was isolated by HPLC analysis and identified as monovinyltripropionic acid porphyrin by mass spectrometry. This monovinylic reaction intermediate exhibited identical chromatographic behavior during HPLC analysis as harderoporphyrin (3-vinyl-8,13,17-tripropionic acid-2,7,12,18-tetramethylporphyrin). Furthermore, HemN was able to utilize chemically synthesized harderoporphyrinogen as substrate and converted it to protoporphyrinogen IX. These results suggest that during HemN catalysis the propionate side chain of ring A of coproporphyrinogen III is decarboxylated prior to that of ring B.

Staphylococcus aureus Coproporphyrinogen III Oxidase Is Required for Aerobic and Anaerobic Heme Synthesis.

The virulence of the human pathogen Staphylococcus aureus is supported by many heme-dependent proteins, including key enzymes of cellular respiration. Therefore, synthesis of heme is a critical component of staphylococcal physiology. S. aureus generates heme via the coproporphyrin-dependent pathway, conserved across members of the Firmicutes and Actinobacteria In this work, we genetically investigate the oxidation of coproporphyrinogen to coproporphyrin in this heme synthesis pathway. The coproporphyrinogen III oxidase CgoX has previously been identified as the oxygen-dependent enzyme responsible for this conversion under aerobic conditions. However, because S. aureus uses heme during anaerobic nitrate respiration, we hypothesized that coproporphyrin production is able to proceed in the absence of oxygen. Therefore, we tested the contribution to anaerobic heme synthesis of CgoX and two other proteins previously identified as potential oxygen-independent coproporphyrinogen dehydrogenases, NWMN_1486 and NWMN_1636. We have found that CgoX alone is responsible for aerobic and anaerobic coproporphyrin synthesis from coproporphyrinogen and is required for aerobic and anaerobic heme-dependent growth. This work provides an explanation for how S. aureus heme synthesis proceeds under both aerobic and anaerobic conditions.IMPORTANCE Heme is a critical molecule required for aerobic and anaerobic respiration by organisms across kingdoms. The human pathogen Staphylococcus aureus has served as a model organism for the study of heme synthesis and heme-dependent physiology and, like many species of the phyla Firmicutes and Actinobacteria, generates heme through a coproporphyrin intermediate. A critical step in terminal heme synthesis is the production of coproporphyrin by the CgoX enzyme, which was presumed to be oxygen dependent. However, S. aureus also requires heme during anaerobic growth; therefore, the synthesis of coproporphyrin by an oxygen-independent mechanism is required. Here, we identify CgoX as the enzyme performing the oxygen-dependent and -independent synthesis of coproporphyrin from coproporphyrinogen, resolving a key outstanding question in the coproporphyrin-dependent heme synthesis pathway.

Isolated Bacillus subtilis HemY has coproporphyrinogen III to coproporphyrin III oxidase activity.

Oxidation of coproporphyrinogen III to coproporphyrin III is found in extracts of Escherichia coli cells containing the Bacillus subtilis HemY protein (M. Hansson and L. Hederstedt, J. Bacteriol. 176, 5962-5970). We have analysed whether this activity is due to the heterologous expression system, since it in vivo would lead to disruption of the heme biosynthetic pathway. B. subtilis hemY was fused in its 3'-end to a polynucleotide encoding six histidine residues and expressed from plasmids in both E. coli and B. subtilis. The His6-tagged HemY protein extracted from membranes using non-ionic detergent was purified by Ni2+ affinity chromatography. Isolated HemY fusion protein synthesised in E. coli and B. subtilis oxidised coproporphyrinogen III to coproporphyrin III. No direct formation of protoporphyrin IX from coproporphyrinogen III could be detected. Our results suggest that the coproporphyrinogen III to coproporphyrin III activity of HemY is either avoided in B. subtilis in vivo or that coproporphyrin III is a heme biosynthetic intermediate in this bacterium.

[Study of the mechanism of the effect of extracellular pH on the synthesis of the oxidative complex (cytochrome a+a3) of Bacillus coagulans: relationship to the "glucose effect" and role of excreted coproporphyrin III (author's transl)]. / Etude du mécanisme de l'influence du pH extracellulaire sur la synthèse du complexe oxydasique (cytochromes a+a3) chez Bacillus coagulans. Relation avec "l'effet glucose" et rôle de la coproporphyrine III excrétée.

During the "respiratory adaptation" of Bacillus coagulans, it was possible to dissociate the kinetics of cytochrome a and a3 synthesis with carbon monoxide. The synthesis of cytochrome a3 is preferentially repressed when the pH of the incubation medium is pH 6.5 instead of pH 5.5. However, though the total synthesis of tetrapyrrole compounds is the same at both pH values, the excretion of coproporphyrin III is much increased at pH 6.5. Bacillus coagulans, sensitive to the "glucose effect", shows the "pH effect" only in the presence of high glucose concentrations. The repression of the oxidase complex synthesis by a slight increase of the extracellular pH appears directly related to the increase of the extracellular coproporphyrin III.

Density-functional study of mechanisms for the cofactor-free decarboxylation performed by uroporphyrinogen III decarboxylase.

Uroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination of carboxyl groups from the four acetate side chains of uroporphyrinogen III to yield coproporphyrinogen III. The enzyme acts by successively protonating each of the four pyrrole rings present in the substrate, thereby allowing decarboxylation of their side chains, but the identity of the proton donors has not been established yet. Tyr164 has been suggested as a proton donor, and Asp86 has been proposed to act either as a proton donor or as an intermediate-stabilizing residue. We have performed density-functional calculations to study this reaction mechanism, and found that the rate-limiting step is substrate protonation, rather than decarboxylation. Surprisingly, whereas Tyr164 is unable to protonate the substrate, this protonation can be effected by a nearby arginine residue (Arg37), with a free energy barrier of 21.4 kcal.mol(-1), in remarkable agreement with the experimental value of 19.5 kcal.mol(-1). The central positioning of this residue in close proximity to all four pyrrole rings in the substrate may play a key role in the sequential activation of each of these moieties.

In situ conversion of coproporphyrinogen to heme by murine mitochondria: terminal steps of the heme biosynthetic pathway.

Coproporphyrinogen oxidase (EC 1.3.3.3), protoporphyrinogen oxidase (EC 1.3.3.4), and ferrochelatase (EC 4.99.1.1) catalyze the terminal three steps of the heme biosynthetic pathway. All three are either bound to or associated with the inner mitochondrial membrane in higher eukaryotic cells. A current model proposes that these three enzymes may participate in some form of multienzyme complex with attendant substrate channeling (Grand-champ, B., Phung, N., & Nordmann, Y., 1978, Biochem. J. 176, 97-102; Ferreira, G.C., et al., 1988, J. Biol. Chem. 263, 3835-3839). In the present study we have examined this question in isolated mouse mitochondria using two experimental approaches: one that samples substrate and product levels during a timed incubation, and a second that follows dilution of radiolabeled substrate by pathway intermediates. When isolated mouse mitochondria are incubated with coproporphyrinogen alone there is an accumulation of free protoporphyrin. When Zn is added as a substrate for the terminal enzyme, ferrochelatase, along with coproporphyrinogen, there is formation of Zn protoporphyrin with little accumulation of free protoporphyrin. When EDTA is added to this incubation mixture with Zn, Zn protoporphyrin formation is eliminated and protoporphyrin is formed. We have examined the fate of radiolabeled substrates in vitro to determine if exogenously supplied pathway intermediates can compete with the endogenously produced compounds. The data demonstrate that while coproporphyrinogen is efficiently converted to heme in vitro when the pathway is operating below maximal capacity, exogenous protoporphyrinogen can compete with endogenously formed protoporphyrinogen in heme production.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic studies of novel di- and tri-propionate substrates for the chicken red blood cell enzyme coproporphyrinogen oxidase.

Coproporphyrinogen oxidase is an important enzyme in heme biosynthesis and catalyses the sequential oxidative decarboxylation of propionates on the A and B rings of the porphyrinogen ring. The effects of substituents on the C and D rings have not been systematically evaluated for their effects on the kinetic constants, K(m) and V(max). A series of synthetic porphyrinogens have been tested for their ability to affect these kinetic constants for the chicken enzyme. The enzyme exhibited the largest V(max) when incubated with the authentic substrate and was clearly able to distinguish between various substituents on the C and D rings of the macrocycle. When co-incubated with substrate, the authentic product, protoporphyrinogen-IX, appears to inhibit coproporphyrinogen oxidase and this may have an important role in the regulation of this enzyme. Thus the model for the active site of this enzyme should be modified to take these factors into account.

Porphyrinurias and occupational disease.

Pathologic porphyrinuria in man is based on a complex etiology and pathogenesis. In hepatic porphyrias, coproporphyrinuria is usually only one of the pathognomostic porphyrin parameters in the urine. Secondary coproporphyrinuria means that an increased excretion of coproporphyrin occurs as the biochemically dominant symptom of a disturbance in porphyrin and heme metabolism during an intoxication, individual condition, or basic disease. Certain foreign and environmental chemicals, such as hexachlorobenzene, polyhalogenated aromatic hydrocarbons, vinyl chloride, and dioxin, alter the heme pathway functionally. Increased porphyrinuria can follow as a toxic response that is differentiated into secondary coproporphyrinuria and chronic hepatic porphyria. This is characterized by a simultaneous increase in hepatic and urinary uroporphyrin and heptacarboxylic porphyrins, owing to inhibition of hepatic uroporphyrinogen decarboxylase. Most of the coproporphyrinurias observed in man are caused by alcohol ingestion. Dioxin, vinyl chloride, and polyhalogenated biphenyls induce an incipient subclinical stage of chronic hepatic porphyria in persons with normal red cell uroporphyrinogen decarboxylase. In contrast, exposure to dioxin on the part of persons with inherited uroporphyrinogen decarboxylase deficiency can cause latent chronic hepatic porphyria to develop into PCT. Coproporphyrinuria and latent chronic hepatic porphyria do not produce clinical symptoms. Secondary porphyrinuria with transition to chronic hepatic porphyria is a metabolic response following various toxic and pathologic conditions; it serves as a sensitive index for chemical exposure and occupational disease.

Oxygen-dependent coproporphyrinogen-III oxidase from Escherichia coli: one-step purification and biochemical characterisation.

Coproporphyrinogen-III oxidase (CPO) catalyses the conversion of coproporphyrinogen-III to protoporphyrinogen-IX in the haem biosynthetic pathway, and its deficient activity is associated with human hereditary coproporphyria. The 47% sequence identity between the oxygen-dependent CPO from Escherichia coli and its human counterpart makes the bacterial enzyme a good model system for structural studies of this disease. Therefore, we overexpressed and purified to homogeneity the oxygen-dependent CPO from E. coli and its selenomethionine derivative fused with a His(6)-tag. Both preparations showed a specific activity of 37500 U mg(-1), had a subunit molecular mass of 35 kDa and behaved as a compact shaped dimer. First crystallisation trials produced plate-shaped diffracting crystals.

Hepatic porphyrias. Current concepts.

Acute intermittent porphyria, variegate porphyria, and hereditary coproporphyria are hepatic porphyrias due to enzyme defects that are inherited as autosomal dominants. Porphyria cutanea tarda is considered an acquired disorder. Similar drugs or circumstances are precipitants of acute attacks in all three inherited hepatic porphyrias. The respective biochemical abnormalities are identifiable by simple, readily available laboratory tests. Management of patients with any of the inherited hepatic porphyrias is directed primarily toward prevention of attacks through avoidance of precipitants and through a diet high in carbohydrate. Therapy for porphyria cutanea tarda includes interdiction of alcohol use and repeated phlebotomy.