Accumulation of protoporphyrin IX in light-sensitive mutants of Escherichia coli.

The accumulation of protoporphyrin IX (Proto IX) in light-sensitive mutants of Escherichia coli was detected by spectrofluorimetry. Fluorescence emission and excitation spectra were recorded from extracts of bacterial cells. Proto IX clearly accumulated in cells with mutations in the visA (hemH) gene but not in the wild-type strain CA274 or in visA mutants that had been rendered light-resistant by introduction of the wild-type visA⁺ gene. Accumulation of Proto IX was also not observed in cells with a mutation in the visB gene. These results confirm the hypothesis that the sensitivity of the visA mutants to light is caused by the abnormal accumulation of Proto IX, a substrate of ferrochelatase, as the result of a genetic defect in the gene for ferrochelatase.     

Effect of glucose on the induction of δ-aminolevulinic acid synthase and ferrochelatase in isolated rat hepatocytes by allylisopropylacetamide

The present work shows that allylisopropylacetamide exerts an inducing effect on δ-aminolevulinic acid synthase and ferrochelatase activities in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the inducing effect produced in both enzymes. Glucose inhibits the induction of δ-aminolevulinic acid synthase and ferrochelatase in this in vitro system. A similar effect was observed with fructose and 2-deoxyglucose. No glucose effect was observed with galactose, mannose, glycerol, pyruvate and lactate. The glucose effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. The simple in vitro method used in the present work promises to be a very useful tool for studies of regulatory mechanisms of porphyrin and heme biosynthesis in hepatocytes under normal and pathological conditions (hepatic porphyrias).     

QM/MM study of the insertion of metal ion into protoporphyrin IX by ferrochelatase

Ferrochelatase catalyzes the metallation of protoporphyrin IX in the terminal step of heme biosynthesis. Mutations in the ferrochelatase gene can lead to the disease erythropoietic porphyria. The catalyzing mechanism of ferrochelatase is still not fully understood. In this paper, we have studied the insertion of Fe²⁺ into the protoporphyrin IX ring by Bacillussubtilis ferrochelatase using combined quantum mechanical and molecular mechanics (QM/MM) calculations. Geometries were optimized at the BP86/6-31G∗ level and energies were calculated at the B3LYP/TZVP level. The overall process involves the stepwise displacement of Glu-264, His-183, and a water molecule from Fe²⁺, and the removal of two protons from the porphyrin ring. The rate-determining step is the cleavage of the bond between the oxygen atom of Glu-264 and Fe²⁺, concomitant with the formation of the first Fe-N bond. It has an energy barrier of 57 kJ mol⁻¹. The porphyrin ring is only slightly distorted in the enzyme active site. The residue Tyr-13 plays a key role for the catalytic process extracting two protons from protoporphyrin IX.     

Crosstalk between metal ions in Bacillus subtilis ferrochelatase

Ferrochelatase (EC 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, catalyzes the insertion of Fe²⁺ into protoporphyrin IX, generating heme. In vitro assays have shown that all characterized ferrochelatases can also incorporate Zn²⁺ into protoporphyrin IX. Previously Zn²⁺ has been observed at an inner metal binding site close to the porphyrin binding site. Mg²⁺, which stimulates Zn²⁺ insertion by Bacillus subtilis ferrochelatase, has been observed at an outer metal binding site. Exchange of Glu272 to a serine eliminated the stimulative effect of Mg²⁺. We found that Zn²⁺ quenched the fluorescence of B. subtilis ferrochelatase and this quenching was used to estimate the metal affinity. Trp230 was identified as the intrinsic fluorophore responsible for the observed quenching pattern. The affinity for Zn²⁺ could be increased by incubating the ferrochelatase with the transition state analogue N-methyl mesoporphyrin IX, which reflected a close collaborative arrangement between the two substrates in the active site. We also showed that the affinity for Zn²⁺ was lowered in the presence of Mg²⁺ and that bound Zn²⁺ was released upon binding of Mg²⁺. In the ferrochelatase with a Glu272Ser modification, the interaction between Zn²⁺ and Mg²⁺ was abolished. It could thereby be demonstrated that the presence of a metal at one metal binding site affected the metal affinity of another, providing the enzyme with a site that regulates the enzymatic activity.     

Regulation of haem biosynthesis in normoblastic erythropoiesis: role of 5-aminolaevulinic acid synthase and ferrochelatase

The development of haem biosynthetic enzyme activity during normoblastic human erythropoiesis was examined in seven patients. The first and last enzymes of the haem biosynthetic pathway, ALA synthase and ferrochelatase, were assayed by radiochemical/high performance liquid chromatographic (HPLC) methods. An assay for ferrochelatase activity in human bone marrow was developed. Enzyme substrates were protoporphyrin IX and ⁵⁹Fe²⁺ ions. ⁵⁹Fe-labelled haem was isolated by organic solvent extraction/sorbent extraction followed by reversed-phase HPLC. Optimal activity occurred at pH 7.3 in the presence of ascorbic acid, in darkness and under anaerobic conditions. Haem production was proportional to cell number and was linear with time to 30 min. The assay was sensitive to the picomolar range of haem production. ALA synthase and ferrochelatase activity was assayed in four highly purified age-matched erythroid cell populations. ALA synthase activity was maximal in the most immature erythoid cells and diminished as the cells matured with an overall five fold loss of activity from proerythroblast to late erythroblast development. Ferrochelatase activity was, however, more stable with less than a two fold change in activity observed during the same period of erythroid differentiation. Maximal activity occurred in erythroid fractions enriched with intermediate erythroblasts. These results support sequential rather than simultaneous appearance of these enzymes during normoblastic erythropoiesis. Quantitative analysis of relative enzyme activity however indicates that at all times during erythroid differentiation ferrochelatase activity is present in excess to that theoretically required relative to ALA synthase activity since ALA and haem are not produced in stoichiometric amounts. The lability of ALA synthase versus the stability and gross relative excess of ferrochelatase activity indicates a far greater role for ALA synthase in the regulation of erythroid haem biosynthesis than for ferrochelatase.     

Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro

Rat liver mitochondrial fractions have previously been shown to contain a pool of iron which was bound neither in cytochromes nor in iron-sulfur centers (Tangerås, A., Flatmark, T., Bäckström, D. and Ehrenberg, A. (1980) Biochim. Biophys. Acta 589, 162–175), and in the present study the availability of this iron pool for heme synthesis has been studied in isolated mitochondria. A minor fraction of this iron is here shown to originate from iron-rich lysosomes present as a contaminant in mitochondrial fractions isolated by differential centrifugation, and a method for the selective quantitation of this iron pool was developed. The availability of the mitochondrial iron pool for heme synthesis by mitochondria in vitro was studied using a recently developed HPLC method for the assay of ferrochelatase activity. When deuteroporphyrin was used as the substrate, 1.04±0.13 nmol/mg protein of deuteroheme was formed after 6 h incubation at 37°C when a plateau was approached, and the initial rate of heme synthesis was 0.3 nmol/h per mg protein. Heme formation from the physiological substrate protoporphyrin was also seen. The heme synthesis increased with the amount of mitochondria used and was blocked by both Fe(II) and Fe(III) chelators. The heme synthesis was independent of mitochondrial oxidizable substrates and no difference was observed between pH 7.4 and 6.5. FMN slightly stimulated the formation of heme from endogenous iron, probably by mobilization of a small amount of contaminating lysosomal iron present in the preparations. The possibility that the mitochondrial iron pool functions as the proximate iron donor for heme synthesis by ferrochelatase in vivo is discussed.     

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 microorganisms 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.     

A novel mutation in erythropoietic protoporphyria: an aberrant ferrochelatase mRNA caused by exon skipping during RNA splicing

An aberrant ferrochelatase mRNA lacking exon 10 was found in a patient with erythropoietic protoporphyria (EPP). In her genomic DNA an A → T transversion at position ?3 of the donor site of intron 10 appeared to be responsible for the exon skipping. Both the patient and her sister were heterozygous for this mutation.     

Biosynthesis of glycoproteins in Candida albicans: Processing of a Man6-GlcNAc2-Asn oligosaccharide by membrane fractions

Abstract Bacillus subtilis can synthesise cytochromes containing a -, b -, c - and d -type heme. The biosynthetic pathways of these heme prosthetic groups were investigated by using strains blocked in uroporphyrinogen III synthesis from porphobilinogen or in heme b (protoheme IX) synthesis from uroporphyrinogen III. The results strongly suggest that heme a and heme d are both synthesised from heme b (protoheme IX). They also indicate that B. subtilis contains a novel ferrochelatase involved in the synthesis of siroheme.     

铁与帕金森病关系研究进展

大量研究证实帕金森患者黑质铁异常沉积,脑内铁代谢紊乱,铁促进氧化应激是神经细胞损伤的重要病理机制。本文综述了铁代谢异常与帕金森病关系的研究概况,指出从中药中寻找金属螯合活性成分治疗PD是极其诱人的研究方向。