Heme Synthase Research Articles

Trapping the substrate radical of heme synthase AhbD.

The heme synthase AhbD catalyzes the last step of the siroheme-dependent heme biosynthesis pathway, which is operative in archaea and sulfate-reducing bacteria. The AhbD-catalyzed reaction consists of the oxidative decarboxylation of two propionate side chains of iron-coproporphyrin III to the corresponding vinyl groups of heme b. AhbD is a Radical SAM enzyme employing radical chemistry to achieve the decarboxylation reaction. Previously, it was proposed that the central iron ion of the substrate iron-coproporphyrin III participates in the reaction by enabling electron transfer from the initially formed substrate radical to an iron-sulfur cluster in AhbD. In this study, we investigated the substrate radical that is formed during AhbD catalysis. While the iron-coproporphyrinyl radical was not detected by electron paramagnetic resonance (EPR) spectroscopy, trapping and visualization of the substrate radical was successful by employing substrate analogs such as coproporphyrin III and zinc-coproporphyrin III. The radical signals detected by EPR were analyzed by simulations based on density functional theory (DFT) calculations. The observed radical species on the substrate analogs indicate that hydrogen atom abstraction takes place at the β-position of the propionate side chain and that an electron donating ligand is located in proximity to the central metal ion of the porphyrin.

The Radical SAM Heme Synthase AhbD from Methanosarcina barkeri Contains Two Auxiliary [4Fe-4S] Clusters.

In archaea and sulfate-reducing bacteria, heme is synthesized via the siroheme-dependent pathway. The last step of this route is catalyzed by the Radical SAM enzyme AhbD and consists of the conversion of iron-coproporphyrin III into heme. AhbD belongs to the subfamily of Radical SAM enzymes containing a SPASM/Twitch domain carrying either one or two auxiliary iron-sulfur clusters in addition to the characteristic Radical SAM cluster. In previous studies, AhbD was reported to contain one auxiliary [4Fe-4S] cluster. In this study, the amino acid sequence motifs containing conserved cysteine residues in AhbD proteins from different archaea and sulfate-reducing bacteria were reanalyzed. Amino acid sequence alignments and computational structural models of AhbD suggested that a subset of AhbD proteins possesses the full SPASM motif and might contain two auxiliary iron-sulfur clusters (AuxI and AuxII). Therefore, the cluster content of AhbD from Methanosarcina barkeri was studied using enzyme variants lacking individual clusters. The purified enzymes were analyzed using UV/Visible absorption and EPR spectroscopy as well as iron/sulfide determinations showing that AhbD from M. barkeri contains two auxiliary [4Fe-4S] clusters. Heme synthase activity assays suggested that the AuxI cluster might be involved in binding the reaction intermediate and both clusters potentially participate in electron transfer.

The auxiliary [4Fe-4S] cluster of the Radical SAM heme synthase from Methanosarcina barkeri is involved in electron transfer.

The heme synthase AhbD catalyzes the oxidative decarboxylation of two propionate side chains of iron-coproporphyrin III to the corresponding vinyl groups of heme during the alternative heme biosynthesis pathway occurring in sulfate-reducing bacteria and archaea. AhbD belongs to the family of Radical SAM enzymes and contains two [4Fe-4S] clusters. Whereas one of these clusters is required for substrate radical formation, the role of the second iron-sulfur cluster is not known. In this study, the function of the auxiliary cluster during AhbD catalysis was investigated. Two single cluster variants of AhbD from M. barkeri carrying either one of the two clusters were created. Using these enzyme variants it was shown that the auxiliary cluster is not required for substrate binding and formation of the substrate radical. Instead, the auxiliary cluster is involved in a late step of AhbD catalysis most likely in electron transfer from the reaction intermediate to a final electron acceptor. Moreover, by using alternative substrates such as coproporphyrin III, Cu-coproporphyrin III and Zn-coproporphyrin III for the AhbD activity assay it was observed that the central iron ion of the porphyrin substrate also participates in the electron transfer from the reaction intermediate to the auxiliary [4Fe-4S] cluster. Altogether, new insights concerning the completely uncharacterized late steps of AhbD catalysis were obtained.

Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants.

Chloroplast signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of plastid-to-nucleus (retrograde) signaling. In Arabidopsis thaliana, genetic studies using norflurazon (NF), an inhibitor of carotenoid biosynthesis, have identified five GUN (genomes uncoupled) genes, implicating the tetrapyrrole pathway as a source of a retrograde signal. Loss of function of any of these GUN genes leads to increased expression of photosynthesis-associated nuclear genes (PhANGs) when chloroplast development has been blocked by NF. Here we present a new Arabidopsis gain-of-function mutant, gun6-1D, with a similar phenotype. The gun6-1D mutant overexpresses the conserved plastid ferrochelatase 1 (FC1, heme synthase). Genetic and biochemical experiments demonstrate that increased flux through the heme branch of the plastid tetrapyrrole biosynthetic pathway increases PhANG expression. The second conserved plant ferrochelatase, FC2, colocalizes with FC1, but FC2 activity is unable to increase PhANG expression in undeveloped plastids. These data suggest a model in which heme, specifically produced by FC1, may be used as a retrograde signal to coordinate PhANG expression with chloroplast development.

Identification of subdomains in NADPH oxidase-4 critical for the oxygen-dependent regulation of TASK-1 K+ channels

Hypoxic inhibition of K+ current is a critical O2-sensing mechanism. Previously, it was demonstrated that the cooperative action of TASK-1 and NADPH oxidase-4 (NOX4) mediated the O2-sensitive K+ current response. Here we addressed the O2-sensing mechanism of NOX4 in terms of TASK-1 regulation. In TASK-1 and NOX4-coexpressing human embryonic kidney 293 cells, hypoxia (5% O2) decreased the amplitude of TASK-1 current (hypoxia-DeltaI(TASK-1)). To examine whether reactive oxygen species (ROS) mediate the hypoxia-DeltaI(TASK-1), we treated the cells with carbon monoxide (CO) which is known to reduce ROS generation from the heme-containing NOX4. Unexpectedly, CO failed to mimic hypoxia in TASK-1 regulation, rather blocked the hypoxia-DeltaI(TASK-1). Moreover, the hypoxia-DeltaI(TASK-1) was neither recovered by H2O2 treatment nor prevented by antioxidant such as ascorbic acid. However, the hypoxia-DeltaI(TASK-1) was noticeably attenuated by succinyl acetone, a heme synthase inhibitor. To further evaluate the role of heme, we constructed and expressed various NOX4 mutants, such as HBD(-) lacking the heme binding domain, NBD(-) lacking the NADPH binding domain, FBD(-) lacking the FAD binding domain, and HFBD(-) lacking both heme and FAD domains. The hypoxia-DeltaI(TASK-1) was significantly reduced in HBD(-)-, FBD(-)-, or HFBD(-)-expressing cells, versus wild-type NOX4-expressing cells. However, NBD(-) did not affect the TASK-1 response to hypoxia. We also found that p22 is required for the NOX4-dependent TASK-1 regulation. These results suggest that O2 binding with NOX4 per se controls TASK-1 activity. In this process, the heme moiety and FBD seem to be responsible for the NOX4 regulation of TASK-1, and p22 might support the NOX4-TASK-1 interaction.

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.

Cytochrome P450 system and heme synthesis enzymes activity in flounder liver as biomarkers of marine environments pollution

The total cytochrome P450 (CYP) levels, aryl hydrocarbon hydroxylase (AHH), aminopyrine N-demethylase (APND), 5-aminolevulinic acid synthase (ALA-S), and heme synthase (HEM-S) activities were analyzed in liver of flounder ( Platichthys flesus) collected from urban and nonurban embayments of the Baltic Sea off Tallinn area to show the response of biomarkers to anthropogenic contaminants, especially carcinogenic PAHs, in marine environments. CYP, APND, ALA-S, and HEM-S values were higher in flounder from the urban sampling site. This correlates well with higher level of PAHs in tissues of flounder from the same site. AHH activities were quite high but have not shown statistically significant site difference although a slight tendency to be higher in flounder liver from the urban embayment was noticed. This study has confirmed the capability of AHH assay to demonstrate toxic contamination of marine environments in relation to the Baltic flounder as well as in previously tested species from other areas. Analyses of APND, ALA-S, and HEM-S activities are recommended as useful biomarkers for evaluation of general toxic load of environmental contamination.

The ferrochelatase gene structure and molecular defects associated with erythropoietic protoporphyria.

Ferrochelatase [heme synthase, protoheme ferrolyase (EC 4.99.1.1)], the terminal enzyme of the heme biosynthetic pathway, catalyzes the incorporation of ferrous ion into protoporphyrin IX to form protoheme IX. The genes and cDNAs for ferrochelatase from mammals and micro-organisms have been isolated. The gene for human ferrochelatase has been mapped to chromosome 18q 21.3 and consists of 11 exons with a size of about 45 kilodaltons. The induction of ferrochelatase expression occurs during erythroid differentiation, and can be attributed to the existence of the promoter sequences of erythroid-related genes. Analysis of the ferrochelatase gene in patients with erythropoietic protoporphyria, an inherited disease caused by ferrochelatase defects, revealed that molecular anomalies of ferrochelatase from 11 patients were found in 9 patients as autosomal dominant type, and 2 patients as recessive type. Diversity of the mutations of the ferrochelatase gene is also briefly described.

Molecular and genetic characterization of ferrochelatase

Ferrochelatase (heme synthase, protoheme ferrolyase [EC 4.99.1.1]), the final enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous ion into protoporphyrin IX to produce protoheme IX. The thorough understanding of the enzyme is prerequisite to elucidating the regulation of iron and heme metabolism. The enzyme's activity is found on the inner mitochondrial membrane of a variety of mammalian cells. The enzyme catalyzes the chelation not only of iron but also of divalent metal ions including cobalt and zinc, and the activity is affected by various metals and lipids. The molecular weights of eukaryotic ferrochelatases are 40,000-42,000 daltons. Complementary DNA (cDNA) encoding ferrochelatase from mouse, human, yeast and bacteria have been isolated, and the derived amino acid sequences show 27-88% homologies among species. The expression of ferrochelatase seems to occur in all living cells, and to play an important role in the regulation of heme biosynthesis. Ferrochelatase is markedly induced at the transcriptional level during erythroid differentiation when iron uptake by cells and hemoglobin synthesis are upregulated. This induction can be explained by the existence of sequences characteristic of erythroid-related genes. The gene has been mapped to human chromosome 18q21.3 and contains 11 exons with a size of about 45 kilobases. Once the gene for human ferrochelatase is cloned, the molecular basis and clinical diagnosis of erythropoietic protoporphyria, caused by a deficiency of ferrochelatase, will become possible. This review summarizes recent advances in ferrochelatase research and suggests important subjects for future research.

Effect of exogenous erythropoietin on haem synthesis in anaemic patients with rheumatoid arthritis.

The effect of recombinant human erythropoietin (rHuEPO) on haem biosynthesis in peripheral red blood cells was evaluated in 12 patients with RA and anaemia (mean haemoglobin concentration 102 g/l, range 90-109 g/l). Before treatment, the serum concentrations of erythropoietin (EPO) were low (mean 13 pmol/l, range 5-32 pmol/l), the activities of haem-synthesizing enzymes within the reference intervals and the erythrocyte protoporphyrin (E-PROTO) concentrations clearly higher than normal. Nine patients responded with an increase in the haemoglobin level of 15 g/l or more. rHuEPO induced a rise in the mean haem synthase (HAEM-S) activity from a baseline of 12.1 to a maximum of 26.8 pmol/h per 10(6) reticulocytes after 20 weeks of treatment (P < 0.002). The mean E-PROTO concentration also rose and reached its maximum at 8 weeks of treatment. We conclude that correction of anaemia in patients with RA using rHuEPO is associated with an activation of HAEM-S, commonly regarded as the rate-limiting enzyme of haem synthesis in erythroid cells. Functional iron deficiency probably explains the simultaneous rise in E-PROTO concentration.

Molecular and genetic characterization of ferrochelatase.

Ferrochelatase (heme synthase, protoheme ferrolyase [EC 4.99.1.1]), the final enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous ion into protoporphyrin IX to produce protoheme IX. The thorough understanding of the enzyme is prerequisite to elucidating the regulation of iron and heme metabolism. The enzyme's activity is found on the inner mitochondrial membrane of a variety of mammalian cells. The enzyme catalyzes the chelation not only of iron but also of divalent metal ions including cobalt and zinc, and the activity is affected by various metals and lipids. The molecular weights of eukaryotic ferrochelatases are 40,000-42,000 daltons. Complementary DNA (cDNA) encoding ferrochelatase from mouse, human, yeast and bacteria have been isolated, and the derived amino acid sequences show 27-88% homologies among species. The expression of ferrochelatase seems to occur in all living cells, and to play an important role in the regulation of heme biosynthesis. Ferrochelatase is markedly induced at the transcriptional level during erythroid differentiation when iron uptake by cells and hemoglobin synthesis are upregulated. This induction can be explained by the existence of sequences characteristic of erythroid-related genes. The gene has been mapped to human chromosome 18q21.3 and contains 11 exons with a size of about 45 kilobases. Once the gene for human ferrochelatase is cloned, the molecular basis and clinical diagnosis of erythropoietic protoporphyria, caused by a deficiency of ferrochelatase, will become possible. This review summarizes recent advances in ferrochelatase research and suggests important subjects for future research.

Hydrogen sulphide poisoning: blood sulphide concentration and changes in haem metabolism.

Hydrogen sulphide is still a potential hazard in several occupations12 and especially in sulphate pulp mills (P J?ppinen, academic dissertation) and oil refineries. Blood sulphide may be used as an indicator of an excessive human hydrogen sulphide exposure. Fatal hydrogen sulphide concentrations are known.34 Blood sulphide concentrations have been determined before by ion selective electrodes combined with Con way microdiffusion cells but only high concentrations, 1-70-3-75 mg/1, in fatal cases could be detected by this method.4 Recently, the method using ion selective electrodes has been improved by preconcentration of trapped sulphide in sodium hydroxide solution.5 This new application allows blood sulphide determinations at low concen trations and it is thus suitable also for non-fatal hydrogen sulphide poisonings. Although decreased activities of haem synthesising enzymes, especially delta-aminolaevulinic acid syn thase (ALA-S) and haem synthase (Haem-S) have been reported to occur in workers exposed to hydrogen sulphide and methyl mercaptan,6 it has been difficult to assess the severity of hydrogen sulphide poisoning in non-fatal cases. The aim of the present work was to evaluate the usefulness of blood sulphide concentrations and activities of haem syn thesising enzymes in this respect.

Erythropoietin, aluminium, and anaemia in patients on haemodialysis

Effects of erythropoietin treatment on haem synthesis in peripheral blood were evaluated in 11 patients on haemodialysis. After 2 weeks of erythropoietin, mean (SEM) uroporphyrinogen-l synthase activity increased significantly from 88 (10) to 116 (9) pmol/h per mg protein. Haem synthase activity, thought to be the rate-limiting step in erythroid haem synthesis, also showed a significant increase from 4.5 (0.8) to 8.4 (1.8) pmol/h per 10(6) reticulocytes. 4 patients, who showed only a partial response to erythropoietin, had significantly higher serum aluminium concentrations than the 7 who responded fully (225 [44] vs 55 [23] micrograms/l); erythrocyte protoporphyrin concentrations in partial responders were also much higher than in responders (973 [120] vs 388 [29] nmol/l). Aluminium intoxication may cause resistance to erythropoietin by interference with haem synthesis, with accumulation of protoporphyrin.

Disturbed iron metabolism among workers exposed to organic sulfides in a pulp plant.

The aim of this study was to investigate a possible relationship between exposure to sulfides and disturbances of the synthesis of heme and the erythrocytes. Eighteen workers exposed to sulfides at a pulp and paper plant were examined and compared with individually matched referents from a thermomechanical pulp plant without such exposure. The exposure levels of methylmercaptan, dimethylsulfide, and dimethyldisulfide were low. However, five subjects were exposed to high levels of short duration, and their data were analyzed separately. The activity of the enzymes delta-aminolevulinic acid synthase and heme synthase in reticulocytes, characteristics of the erythrocytes, and the iron status were analyzed. A minor decrease, not statistically significant, was observed for the enzymes among the five highly exposed subjects. However, the concentrations of iron and transferrin were elevated and the concentration of ferritin was low in comparison to the corresponding levels of the referents. This combination will not occur spontaneously. A previous study indicated that sulfides may inhibit heme synthesis, and the present study suggests that they may also disturb iron metabolism.

Heme arginate treatment for myelodysplastic syndromes

Heme arginate was given to 26 patients with a myelodysplastic syndrome (MDS) as infusions of 2–3 mg/kg body weight weekly for 8–12 weeks. Most of the patients first received a loading dose on four consecutive days. Six of the patients showed improvement in cytopenias during the therapy. In three of the responders severely depressed blood cell counts recovered to normal or close to normal. So far the maximum duration of a response after the cessation of the treatment is 25 months, and the two ongoing responses have lasted for 11 and 12 months, respectively. In two responders of the eight patients with more than 15% ring sideroblasts the number of ring sideroblasts decreased during the treatment but remained unchanged in six non-responders. The responders were characterized by a low or low normal heme synthase activity which increased during the treatment, whereas the non-responders showed a higher mean heme synthase activity which decreased during the treatment. In general, the responders had significantly fewer defects in heme synthetic enzyme activities than the non-responders. FAB type, karyotype or growth pattern in in vitro cultures of hematopoietic progenitors did not predict the response. Apart from one case of mild venous irritation, no other adverse effects were seen. The present study shows that heme arginate induces beneficial effects on cytopenia in some MDS patients and has very few side-effects.

Effects of repeated intravenous administration of haem arginate upon hepatic metabolism of foreign compounds in rats and dogs.

Haem arginate is a new haem compound, recently introduced for the treatment of acute hepatic porphyrias. Porphyrias are characterized biochemically by decreased formation of haem due to defects in certain enzyme activities involved in the haem biosynthesis. Haem is essential for cell respiration and oxidative biotransformation. Hepatic drug metabolism, haem biosynthesis and catabolism were investigated after repeated intravenous administration of haem arginate in connection with toxicity studies. The daily doses of haem for rats were 4, 12 and 40 mg kg-1 and for dogs 3 and 9 mg kg-1 for 30 days and for 28 days, respectively. Hepatic microsomes were used in the assay of the following drug metabolizing enzymes: cytochrome P-450 and b5, aminopyrine N-demethylase, ethoxyresorufin O-deethylase and UDP-glucuronyl transferase. The assay of NADPH-cytochrome C-reductase and the enzymes reflecting synthesis and metabolism of haem in the liver (delta-aminolaevulinic acid synthase, delta-aminolaevulinic acid dehydratase, uroporphyrinogen I-synthase, uroporphyrinogen decarboxylase, haem synthase, haem oxygenase and biliverdin reductase) were performed from 20,000 g supernatants. The lowest dose administered to rats and dogs did not cause any significant changes compared to controls in the parameters measured. The highest doses significantly increased the activities of haem oxygenase and uroporphyrinogen I-synthase but decreased concentrations or activities of other enzymes, e.g. cytochrome P-450 and ethoxyresorufin O-deethylase. The results show that it is important to avoid overdosage of haem when restoration of mixed function oxygenase activity is needed.

Fatal liver failure in protoporphyria: Synergism between ethanol excess and the genetic defect

Protoporphyria was diagnosed in a 56-yr-old man based upon a typical clinical and family history, marked increases in erythrocyte and fecal protoporphyrin concentrations, and a marked decrease (21% of normal) in activity of hepatic heme synthase. Routine tests of liver function and histology were normal, except for a slight increase in bromsulphalein retention (9% at 45 min). Liver chemistries remained normal for 8 more years, but deteriorated rapidly when the patient was 63 yr old, with cholestasis precipitated by injury due to excess intake of ethanol. This, in turn, led to a defect in hepatic protoporphyrin excretion and to further worsening of liver injury due to porphyrin deposition. Our patient represents the 21st and oldest patient thus far reported to have died of liver failure complicating protoporphyria.

Conversion of 5-aminolaevulinate into haem by homogenates of human liver. Comparison with rat and chick-embryo liver homogenates

To assess whether the synthesis of haem can be studied in small amounts of human liver, we measured kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of human livers. We used methods previously developed in our laboratory for studies of rat and chick-embryo livers [Healey, Bonkowsky, Sinclair & Sinclair (1981) Biochem. J. 198, 595-604]. The maximal rate at which homogenates of human livers converted 5-aminolaevulinate into protoporphyrin was only 26% of that for rat, and 58% of that for chick embryo. In the absence of added Fe2+, homogenates of fresh human liver resembled those of chick embryos in that protoporphyrin and haem accumulated in similar amounts, whereas fresh rat liver homogenate accumulated about twice as much haem as protoporphyrin. However, when Fe2+ (0.25 mM) was added to human liver homogenates, mainly haem accumulated, indicating that the supply of reduced iron limited the activity of haem synthase, the final enzyme in the haem-biosynthesis pathway. Addition of the potent iron chelator desferrioxamine after 30 min of incubation with 5-amino[14C]laevulinate stopped further haem synthesis without affecting synthesis of protoporphyrin. Thus the prelabelled haem was stable after addition of desferrioxamine. Since the conversion of 5-amino[14C]laevulinate into haem and protoporphyrin was carried out at pH 7.4, whereas the pH optimum for rat or bovine hepatic 5-aminolaevulinate dehydratase is about 6.3, we determined kinetic parameters of the human hepatic dehydrase at both pH values. The Vmax was the same at both pH values, whereas the Km was slightly higher at the lower pH. Our results indicate that the synthesis of porphyrins and haem from 5-aminolaevulinate can be studied with the small amounts of human liver obtainable by percutaneous needle biopsy. We discuss the implications of our results in relation to use of rat or chick-embryo livers as experimental models for the biochemical features of human acute porphyria.

Trinitrotoluene-induced effects on rat heme metabolism

A single injection (100 mg/kg body wt) of trinitrotoluene in rats caused decreased δ-aminolevulinic adic synthase activity in reticulocytes and decreased erythrocyte coproporphyrin concentration 48 hr after the intraperitoneal dosage. For comparison, liver δ-aminolevulinic acid synthase was unaffected while heme synthase activity was below the control range at the same time. Heme oxygenase activity increased simultaneously. These effects are likely to lead to a negative heme balance and may be developed for a biological exposure test to trinitrotoluene in occupational health service.

Enzymatic formation of zinc-protoporphyrin by rat liver and its potential effect on hepatic heme metabolism

The mitochondrial enzyme heme synthase (ferrochelatase) catalyzes the formation of heme from ferrous iron and protoporphyrin. Using a fluorometric assay, the enzymatic formation of zinc-protoporphyrin in rat liver tissue was compared with heme formation. With sonicated liver homogenate, the rate of zinc-protoporphyrin formation was similar to that of heme when each metal was present alone in the reaction mixture. When combined in the reaction mixture, the two metals competed for the enzyme. With intact mitochondria, zinc was a better substrate, as 90% of the product was zinc-protoporphyrin in the presence of 50 μM zinc and 50 μM iron. The effect of zinc-protoporphyrin on the induction of δ-aminolevulinic acid synthase, the rate-limiting enzyme in hepatic heme biosynthesis, was also examined in the rat. Intraperitoneal injection of allylisopropylacetamide (350 μmol) induced activity of the enzyme fourfold. Concomitant administration of zinc-protoporphyrin (4 μmol) suppressed the induction by 52%, nearly as effectively as the same amount of heme. These findings indicate that the formation of zinc-protoporphyrin in liver may (a) inhibit the synthesis of heme and (b) exert negative feedback regulation on δ-aminolevulinic acid synthase. This combination would reduce the hepatic heme level.