Bovine ferrochelatase. Kinetic analysis of inhibition by N-methylprotoporphyrin, manganese, and heme

The terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase EC 4.99.1.1), has been purified to apparent homogeneity from bovine liver mitochondria using a scheme similar to that reported by Taketani and Tokunaga (Taketani, S. and Tokunaga, R. (1981) J. Biol. Chem. 256, 12748-12753) for purification of the enzyme from rat liver. The final yield was 49% with a 2000-fold purification. Ferrochelatase has an apparent molecular weight of approximately 40,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and column chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. The purified enzyme was only slightly stimulated by added lipid and was inhibited by Mn2+, Pb2+, and Hg2+. Bovine ferrochelatase utilized proto-, meso-, and deuteroporphyrin, but not disubstituted porphyrins (2,4-disulfonic and 2,4-bisglycol deuteroporphyrin). N-Methylprotoporphyrin, a toxic by-product of the metabolism of some drugs, was found to inhibit ferrochelatase in a competitive fashion with respect to porphyrin with a Ki of 7 nM and uncompetitive with respect to iron. Manganese inhibits ferrochelatase competitively with respect to iron (Ki = 15 microM) and noncompetitively with respect to the porphyrin substrate. Heme, one of the products, is a noncompetitive inhibitor with respect to iron. These findings lead to a sequential Bi Bi kinetic model for ferrochelatase with iron binding occurring prior to porphyrin binding and heme being released prior to the release of two protons.     

Biosynthesis of delta-aminolevulinic acid and the regulation of heme formation by immature erythroid cells in man

Heme formation in the erythron is subject to end product regulation by negative feedback, but the exact point of metabolic control in human erythroid cells is unknown. To investigate the mode of action of heme on its own formation, the effects of micromolar concentrations of hemin on de novo synthesis of protoporphyrin IX and delta-aminolevulinate (delta-ALA) by intact human reticulocytes were examined in the presence of 1 mM alpha,alpha'-bipyridyl and 200 microM 4,6-dioxoheptanoate to block their further conversion by ferrochelatase or delta-ALA dehydrase, respectively. At final concentrations (25-40 microM), hemin, which is known to reduce incorporation of [2-14C]glycine into cellular heme, significantly inhibited formation of protoporphyrin IX and total delta-aminolevulinate in situ by these cells. Since synthesis of the first committed precursor, delta-aminolevulinate, as well as protoporphyrin (which is derived from it) were diminished, the effects of hemin on delta-aminolevulinate synthase (EC 2.3.1.37) were studied. Hemin, at concentrations up to 40 microM, had no direct effect on enzymatic activity, as measured with [5-14C] alpha-ketoglutarate (in hypotonically lysed cells) or [1,4-14C]succinyl coenzyme A (in deoxycholate lysates), even after preincubation. However, when intact human reticulocytes were incubated with hemin before assay for delta-ALA synthase, there was a rapid, concentration-dependent reduction in enzymatic activity (mean 42 and 23% inhibition after 60 min for these two substrates, respectively). Hemin had no effect on steady-state levels of delta-ALA synthase mRNA, as determined by Northern blot hybridization using an erythroid-specific human cDNA probe. Thus, a mechanism for inducing feedback inhibition of the tetrapyrrole pathway exists in human erythroid cells. It controls formation of the first committed precursor of protoporphyrin IX, delta-aminolevulinate, and hence regulates heme biosynthesis by limiting the availability of the porphyrin, rather than the metal substrate for the ferrochelatase reaction. Hemin interacts with constituents of the intact reticulocyte significantly to reduce delta-aminolevulinic acid synthase activity by an indirect cellular process that does not influence the abundance of erythroid-specific synthase mRNA but may either inhibit its ribosomal translation in an unknown manner or promote degradation of the enzyme itself by specific proteolysis.     

Roles of porphyrins and host iron transport proteins in regulation of growth of Porphyromonas gingivalis W50.

Porphyromonas gingivalis (Bacteroides gingivalis) requires iron in the form of hemin for growth and virulence in vitro, but the contributions of the porphyrin ring structure, porphyrin-associated iron, host hemin-sequestering molecules, and host iron-withholding proteins to its survival are unknown. Therefore, the effects of various porphyrins, host iron transport proteins, and inorganic iron sources on the growth of P. gingivalis W50 were examined to delineate the various types of iron molecules used for cellular metabolism. Cell envelope-associated hemin and iron stores contributed to the growth of P. gingivalis in hemin-free culture, and depletion of these endogenous reserves required eight serial transfers into hemin-free medium for total suppression of growth. Comparable growth of P. gingivalis was observed with 7.7 microM equivalents of hemin as hemoglobin (HGB), methemoglobin, myoglobin, hemin-saturated serum albumin, lactoperoxidase, cytochrome c, and catalase. Unrestricted growth was recorded in the presence of haptoglobin-HGB and hemopexin-hemin complexes, indicating that these host defense proteins do not sequester HGB and hemin from P. gingivalis. The iron chelator 2,2'-bipyridyl functionally chelated hemin-associated iron, resulting in dose-dependent inhibition of growth in hemin-restricted cultures at 1 to 25 microM 2,2'-bipyridyl concentrations. In the absence of an exogenous iron source, protoporphyrin IX did not support P. gingivalis growth. These findings suggest that the iron atom in the hemin molecule is the critical constituent for growth and that the tetrapyrrole porphyrin ring structure may represent an important vehicle for delivery of iron into the P. gingivalis cell. P. gingivalis does not have a strict requirement for porphyrins, since growth occurred with nonhemin iron sources, including high concentrations (200 muM) of ferric, ferrous, and nitrogenous inorganic iron, and P. gingivalis exhibited unrestricted growth in the presence of host transferrin, lactoferrin, and serum albumin. The diversity of iron substrates utilized by P. gingivalis and the observation that growth was not affected by the bacteriostatic effects of host iron-withholding proteins, which it may encounter in the periodontal pocket, may explain why P. gingivalis is such a formidable pathogen in the periodontal disease process.     

Effects of hemin on rat liver cyclic AMP-dependent protein kinases in cell extracts and intact hepatocytes

Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 microM hemin and 100 microM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 microM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40-60 microM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 microM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 microM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases.     

The conserved active-site loop residues of ferrochelatase induce porphyrin conformational changes necessary for catalysis

Binding of porphyrin to murine ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is investigated by employing a set of variants harboring mutations in a putative porphyrin-binding loop. Using resonance Raman (RR) spectroscopy, the structural properties of the ferrochelatase-bound porphyrins are examined, especially with respect to the porphyrin deformation occurring in the environment of the active site. This deformation is thought to be a key step in the enzymatic insertion of ferrous iron into the porphyrin ring to make heme. Our previous RR spectroscopic studies of binding of porphyrin to murine ferrochelatase led us to propose that the wild-type enzyme induces porphyrin distortion even in the absence of the metal ion substrate. Here, we broaden this view by presenting evidence that the degree of a specific nonplanar porphyrin deformation contributes to the catalytic efficiency of ferrochelatase and its variants. The results also suggest that the conserved Trp256 (murine ferrochelatase numbering) is partially responsible for the observed porphyrin deformation. Binding of porphyrin to the ferrochelatase variants causes a decrease in the intensity of RR out-of-plane vibrational mode gamma(15), a saddling-like mode that is strong in the wild-type enzyme. In particular, the variant with a catalytic efficiency 1 order of magnitude lower than that of the wild-type enzyme is estimated to produce less than 30% of the wild-type saddling deformation. These results suggest that specific conserved loop residues (especially Trp256) are directly involved in the saddling of the porphyrin substrate.     

Deuteroporphyrin-albumin binding equilibrium. The effects of porphyrin self-aggregation studied for the human and the bovine proteins.

The binding equilibrium of deuteroporphyrin IX to human serum albumin and to bovine serum albumin was studied, by monitoring protein-induced changes in the porphyrin fluorescence and taking into consideration the self-aggregation of the porphyrin. To have control over the latter, the range of porphyrin concentrations was chosen to maker dimers (non-covalent) the dominant aggregate. Each protein was found to have one high-affinity site for deuteroporphyrin IX monomers, the magnitudes of the equilibrium binding constants (25 degrees C, neutral pH, phosphate-buffered saline) being 4.5 (+/- 1.5) X 10(7) M-1 and 1.7 (+/- 0.2) X 10(6) M-1 for human serum albumin and for bovine serum albumin respectively. Deuteroporphyrin IX dimers were found to bind directly to the protein, each protein binding one dimer, with high affinity. Two models are proposed for the protein-binding of porphyrin monomers and dimers in a porphyrin system having both species: a competitive model, where each protein molecule has only one binding site, which can be occupied by either a monomer or a dimer; a non-competitive model, where each protein molecule has two binding sites, one for monomers and one for dimers. On testing the fit of the data to the models, an argument can be made to favour the non-competitive model, the equilibrium binding constants of the dimers, for the non-competitive model (25 degrees C, neutral pH, phosphate-buffered saline), being: 8.0 (+/- 1.8) X 10(8) M-1 and 1.2 (+/- 0.6) X 10(7) M-1 for human serum albumin and bovine serum albumin respectively.     

Interaction of hemin with placental glutathione transferase

To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport.     

The importance of porphyrin distortions for the ferrochelatase reaction

Ferrochelatase is the terminal enzyme in haem biosynthesis, i.e. the enzyme that inserts a ferrous ion into the porphyrin ring. Suggested reaction mechanisms for this enzyme involve a distortion of the porphyrin ring when it is bound to the enzyme. We have examined the energetics of such distortions using various theoretical calculations. With the density functional B3LYP method we calculate how much energy it costs to tilt one of the pyrrole rings out of the porphyrin plane for an isolated porphyrin molecule without or with a divalent metal ion in the centre of the ring. A tilt of 30 degrees costs 65-130 kJ/mol for most metal ions, but only approximately 48 kJ/mol for free-base (neutral) porphine. This indicates that once the metal is inserted, the porphyrin becomes stiffer and flatter, and therefore binds with lower affinity to a site designed to bind a distorted porphyrin. This would facilitate the release of the product from ferrochelatase. This proposal is strengthened by the fact that the only tested metal ion with a lower distortion energy than free-base porphyrin (Cd(2+)) is an inhibitor of ferrochelatase. Moreover, it costs even less energy to tilt a doubly deprotonated porphine(2-) molecule. This suggests that the protein may lower the acid constant of the pyrrole nitrogen atoms by deforming the porphyrin molecule. We have also estimated the structure of the protoporphyrin IX substrate bound to ferrochelatase using combined quantum chemical and molecular mechanics calculations. The result shows that the protein may distort the porphyrin by approximately 20 kJ/mol, leading to a distinctly non-planar structure. All four pyrrole rings are tilted out of the porphyrin mean plane (1-16 degrees ) but most towards the putative binding site of the metal ion. The predicted tilt is considerably smaller than that observed in the crystal structure of a porphyrin inhibitor.     

Metal binding to Saccharomyces cerevisiae ferrochelatase

Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway. It catalyzes the insertion of ferrous iron into protoporphyrin IX to produce protoheme IX. The crystal structures of ferrochelatase from Saccharomyces cerevisiae in free form, in complex with Co(II), a substrate metal ion, and in complex with two inhibitors, Cd(II) and Hg(I), are presented in this work. The enzyme is a homodimer, with clear asymmetry between the monomers with regard to the porphyrin binding cleft and the mode of metal binding. The Co(II) and Cd(II) complexes reveal the metal binding site which consists of the invariant amino acids H235, E314, and S275 and solvent molecules. The shortest distance to the metal reveals that amino acid H235 is the primary metal binding residue. A second site with bound Cd(II) was found close to the surface of the molecule, approximately 14 A from H235, with E97, H317, and E326 participating in metal coordination. It is suggested that this site corresponds to the magnesium binding site in Bacillus subtilis ferrochelatase. The latter site is also located at the surface of the molecule and thought to be involved in initial metal binding and regulation.     

Kinetic studies of human liver ferrochelatase. Role of endogenous metals

Ferrochelatase activity in human liver has been extensively characterized in the mitochondrial fraction by kinetic study of the enzyme in initial velocity conditions. We found that human liver mitochondrial membranes contain large amounts of endogenous metals that are substrates for the enzyme, leading to a lack of linearity of the activity as function of protein concentration. This lack of linearity is mainly due to a high zinc-chelatase activity with endogenous zinc. Under optimal experimental conditions, the maximum velocity for iron incorporation was 8.7 nmol of protoheme/h/mg of protein, and the maximum velocity for zinc incorporation was 4.3 nmol of zinc-protoporphyrin/h/mg of protein. The Michaelis constant for protoporphyrin IX was (i) dependent on the amount of protein when the overall chelatase reactions were measured but (ii) independent of the amount of protein when only zinc-chelatase activity was measured (Km = 0.5 microM). The Michaelis constants for iron and zinc were 0.35 and 0.08 microM, respectively, and the inhibitory constants for competitive incorporation of iron and zinc were KIFe/Zn = 0.12 microM and KIZn/Fe = 0.58 microM. The affinity of the enzyme for zinc lowers the actual determination of ferrochelatase activity with iron as substrate. Furthermore, when measuring ferrochelatase (e.g. in liver biopsy), endogenous zinc content in the biological sample must be taken into account.     

Haematoporphyrin and OO''-diacetylhaematoporphyrin binding by serum and cellular proteins. Implications for the clearance of these photochemotherapeutic agents by cells.

Haematoporphyrin derivative (HpD), a mixture of porphyrins, is currently used as a photochemotherapeutic agent in the treatment of neoplasias. The interaction of purified components of HpD with serum and cellular proteins was investigated using absorption and fluorescence spectroscopy. The interactions of haematoporphyrin and OO'-diacetylhaematoporphyrin with human albumin and with haemopexin, the two major serum porphyrin-binding proteins, show stoichiometries of 1 mol of porphyrin bound per mol of protein. The apparent dissociation constants, Kd, are in the range of 1-2 microM for albumin and 3-4 microM for haemopexin. These two major components of HpD would, after intravenous injection, bind to albumin and circulate in serum as albumin complexes. Free porphyrin rather than porphyrin bound to albumin interacts with Morris hepatoma tissue culture cells. A rapid high-affinity saturable transport system operates at free porphyrin concentrations of less than 2 microM. In addition, fluorescence spectra show that components in rat liver cytosol can bind haematoporphyrin and OO'-diacetylhaematoporphyrin and distinguish these binders from those present in rat serum.     

Modulation of inhibition of ferrochelatase by N-methylprotoporphyrin

Protoporhyrin IX ferrochelatase catalyses the terminal step of the haem-biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. NMPP (N-methylprotoporphyrin), a transition-state analogue and potent inhibitor of ferrochelatase, is commonly used to induce haem deficiency in mammalian cell cultures. To create ferrochelatase variants with different extents of tolerance towards NMPP and to understand further the mechanism of ferrochelatase inhibition by NMPP, we isolated variants with increased NMPP resistance, bearing mutations in an active-site loop (murine ferrochelatase residues 248-257), which was previously shown to mediate a protein conformational change triggered by porphyrin binding. The kinetic mechanisms of inhibition of two variants, in which Pro255 was replaced with either arginine (P255R) or glycine (P255G), were investigated and compared with that of wild-type ferrochelatase. While the binding affinity of the P255X variants for NMPP decreased by one order of magnitude in relation to that of wild-type enzyme, the inhibition constant increased by approximately two orders of magnitude (K(i)(app) values of 1 microM and 2.3 microM for P255R and P255G respectively, as against 3 nM for wild-type ferrochelatase). Nonetheless, the drastically reduced inhibition of the variants by NMPP was not paralleled with a decrease in specificity constant (kcat/K(m, protoporhyrin IX)) and/or catalytic activity (kcat). Further, although NMPP binding to either wild-type ferrochelatase or P255R occurred via a similar two-step kinetic mechanism, the forward and reverse rate constants associated with the second and rate-limiting step were comparable for the two enzymes. Collectively, these results suggest that Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase.     

Nickel(II) chelatase variants directly evolved from murine ferrochelatase: porphyrin distortion and kinetic mechanism

The heme biosynthetic pathway culminates with the ferrochelatase-catalyzed ferrous iron chelation into protoporphyrin IX to form protoheme. The catalytic mechanism of ferrochelatase has been proposed to involve the stabilization of a nonplanar porphyrin to present the pyrrole nitrogens to the metal ion substrate. Previously, we hypothesized that the ferrochelatase-induced nonplanar distortions of the porphyrin substrate impose selectivity for the divalent metal ion incorporated into the porphyrin ring and facilitate the release of the metalated porphyrin through its reduced affinity for the enzyme. Using resonance Raman spectroscopy, the structural properties of porphyrins bound to the active site of directly evolved Ni(2+)-chelatase variants are now examined with regard to the mode and extent of porphyrin deformation and related to the catalytic properties of the enzymes. The Ni(2+)-chelatase variants (S143T, F323L, and S143T/F323L), which were directly evolved to exhibit an enhanced Ni(2+)-chelatase activity over that of the parent wild-type ferrochelatase, induced a weaker saddling deformation of the porphyrin substrate. Steady-state kinetic parameters of the evolved variants for Ni(2+)- and Fe(2+)-chelatase activities increased compared to those of wild-type ferrochelatase. In particular, the reduced porphyrin saddling deformation correlated with increased catalytic efficiency toward the metal ion substrate (Ni(2+) or Fe(2+)). The results lead us to propose that the decrease in the induced protoporphyrin IX saddling mode is associated with a less stringent metal ion preference by ferrochelatase and a slower porphyrin chelation step.     

Association of hemin with protein 4.1 as compared to spectrin and actin

The interaction of hemin with protein 4.1 isolated from red cell membrane cytoskeleton has been studied. Spectrophotometric titration has shown one strong binding site and additional lower affinity sites for hemin. From fluorescence quenching data an association binding constant of 1.3 . 10(7) M-1 has been calculated for the primary site. The conformation of cytoskeletal proteins after hemin binding was followed by the use of far UV circular dichroism and compared to that of the serum hemin trap, albumin. The secondary structure of albumin was unchanged in the presence of high hemin concentrations. Both spectrin and actin lost their conformation upon hemin binding in a ligand-concentration and time-dependent manner. Unlike spectrin and actin, the secondary structure of protein 4.1 appeared. The findings of this study suggest that protein 4.1 may serve as the cytoskeletal temporary sink for small amounts of membrane-intercalated hemin similarly to the function of albumin in the serum. However, an increased release of hemin under pathological conditions may cause hemin association with the cytoskeletal proteins and as a result the cell membrane is expected to be distorted.     

Metal binding to<Emphasis Type="Italic"> Bacillus subtilis</Emphasis> ferrochelatase and interaction between metal sites

Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyses metal insertion into protoporphyrin IX. The location of the metal binding site with respect to the bound porphyrin substrate and the mode of metal binding are of central importance for understanding the mechanism of porphyrin metallation. In this work we demonstrate that Zn(2+), which is commonly used as substrate in assays of the ferrochelatase reaction, and Cd(2+), an inhibitor of the enzyme, bind to the invariant amino acids His183 and Glu264 and water molecules, all located within the porphyrin binding cleft. On the other hand, Mg(2+), which has been shown to bind close to the surface at 7 A from His183, was largely absent from its site. Activity measurements demonstrate that Mg(2+) has a stimulatory effect on the enzyme, lowering K(M) for Zn(2+) from 55 to 24 micro M. Changing one of the Mg(2+) binding residues, Glu272, to serine abolishes the effect of Mg(2+). It is proposed that prior to metal insertion the metal may form a sitting-atop (SAT) complex with the invariant His-Glu couple and the porphyrin. Metal binding to the Mg(2+) site may stimulate metal release from the protein ligands and its insertion into the porphyrin.     

Binding of protoporphyrin IX and metal derivatives to the active site of wild-type mouse ferrochelatase at low porphyrin-to-protein ratios

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe(2+) chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.     

Estrogen synthetase stimulation by hemin in human choriocarcinoma cell culture

The ability of hemin to stimulate estrogen synthetase (aromatase) in cultured human trophoblast cells and in cellular homogenates was investigated and compared with aromatase stimulation by dibutyryl cAMP [(Bu)2 cAMP]. Cells grown with hemin for 24 h, or homogenates incubated for 45 min with hemin, showed maximal aromatase stimulation (150 to 200% of activities in the absence of hemin) at 25 microM and 0.1 microM, respectively. Aromatase stimulation in culture by 25 microM hemin was observed within 4 h after hemin addition, while (Bu)2 cAMP required more than 6 h. Intracellular heme and porphyrin levels were higher (160 to 185%) in 96 h (Bu)2 cAMP-grown cells than control cells.     

Metallation of the transition-state inhibitor N-methyl mesoporphyrin by ferrochelatase: implications for the catalytic reaction mechanism

Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferro-chelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to non-catalytic metallation of N-MeMP, the ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the K(m) values for Zn(II) (17 microM) and Cu(II) (170 microM) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.     

Ferrochelatase from Rhodopseudomonas sphaeroides: substrate specificity and role of sulfhydryl and arginyl residues.

Purified ferrochelatase (protoheme ferrolyase; EC 4.99.1.1) from the bacterium Rhodopseudomonas sphaeroides was examined to determine the roles of cationic and sulfhydryl residues in substrate binding. Reaction of the enzyme sulfhydryl residues with N-ethylmaleimide or monobromobimane resulted in a rapid loss of enzyme activity. Ferrous iron, but not porphyrin substrate, had a protective effect against inactivation by these two reagents. Quantitation with 3H-labeled N-ethylmaleimide revealed that inactivation required one to two sulfhydryl groups to be modified. Modification of arginyl residues with either 2,3-butanedione or camphorquinone 10-sulfonate resulted in a loss of ferrochelatase activity. A kinetic analysis of the modified enzyme showed that the Km for ferrous iron was not altered but that the Km for the porphyrin substrate was increased. These data suggested that arginyl residues may be involved in porphyrin binding, possibly via charge pair interactions between the arginyl residue and the anionic porphyrin propionate side chain. Modification of lysyl residues had no effect on enzyme activity. We also examined the ability of bacterial ferrochelatase to use various 2,4-disubstituted porphyrins as substrates. We found that 2,4-bis-acetal- and 2,4-disulfonate deuteroporphyrins were effective substrates for the purified bacterial enzyme and that N-methylprotoporphyrin was an effective inhibitor of the enzyme. Our data for the ferrochelatase of R. sphaeroides are compared with previously published data for the eucaryotic enzyme.     

Human serum albumin: microheterogeneity and hemin-binding ability]

Microheterogeneity of human serum albumin was studied by isoelectric focussing in ampholines and the borax-borate buffer-mannite system within the pH range 4.0--6.0 and by fractional precipitation in 3 M KCl. Albumin was found to have the same degree of heterogeneity in all separating systems. However, during focussing in ampholines microheterogeneity is partially due to the albumin binding with the ampholines. In other systems similar binding was not observed. Studies of the albumin-hemin complex showed that different fractions bind hemin in a different degree, maximal binding being observed within the pH range of 5.0--5.2. Defatting does not affect the distribution of hemin within the fractions of serum albumin.