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Characterization of the Iron-binding Site in Mammalian Ferrochelatase by Kinetic and Mössbauer Methods

1995; Elsevier BV; Volume: 270; Issue: 44 Linguagem: Inglês

10.1074/jbc.270.44.26352

1083-351X

Ricardo Franco, José J. G. Moura, Isabel Moura, Steven G. Lloyd, Boi Hanh Huynh, William S. Forbes, Glória C. Ferreira,

Hemoglobin structure and function

All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX. Kinetic methods and Mössbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mössbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate. Mössbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of dithiothreitol shows a somewhat broad quadrupole doublet. Spectral analysis indicates that when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the protein. The spectroscopic parameters for this bound species are d = 1.36 ± 0.03 mm/s and ΔEQ = 3.04 ± 0.06 mm/s, distinct from the larger ΔEQ of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with regard to the mechanism of ferrochelatase activity are discussed. All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX. Kinetic methods and Mössbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mössbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate. Mössbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of dithiothreitol shows a somewhat broad quadrupole doublet. Spectral analysis indicates that when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the protein. The spectroscopic parameters for this bound species are d = 1.36 ± 0.03 mm/s and ΔEQ = 3.04 ± 0.06 mm/s, distinct from the larger ΔEQ of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with regard to the mechanism of ferrochelatase activity are discussed. INTRODUCTIONFerrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme of the heme biosynthetic pathway(1Ferreira G.C. Franco R. Lloyd S.G. Moura I. Moura J.J.G. Huynh B.H. J. Bioenerg. Biomembr. 1995; 27: 221-229Crossref PubMed Scopus (56) Google Scholar, 2Dailey H.A. Dailey H.A. Biosynthesis of Heme and Chlorophylls. McGraw-Hill, New York1990: 123-161Google Scholar, 3Jordan P.M. Mgbeje B.I.A. Jordan P.M. Biosynthesis of Tetrapyrroles. Elsevier Science Publishers Ltd., London1991: 257-294Google Scholar). Its function is to catalyze the chelation of ferrous ion into protoporphyrin IX to form protoheme(1Ferreira G.C. Franco R. Lloyd S.G. Moura I. Moura J.J.G. Huynh B.H. J. Bioenerg. Biomembr. 1995; 27: 221-229Crossref PubMed Scopus (56) Google Scholar, 2Dailey H.A. Dailey H.A. Biosynthesis of Heme and Chlorophylls. McGraw-Hill, New York1990: 123-161Google Scholar). Although these are the only physiological substrates, the enzyme is capable of utilizing several other divalent transition metals (e.g. Co2+ and Zn2+) (4Camadro J.-M. Ibraham N.G. Levere R.D. J. Biol. Chem. 1984; 259: 5678-5682Abstract Full Text PDF PubMed Google Scholar, 5Jones M.S. Jones O.T.G. Biochem. J. 1969; 113: 507-514Crossref PubMed Scopus (151) Google Scholar, 6Camadro J.-M. Labbe P. Biochim. Biophys. Acta. 1982; 707: 280-288Crossref PubMed Scopus (33) Google Scholar) and a wide variety of IX isomer porphyrins (2Dailey H.A. Dailey H.A. Biosynthesis of Heme and Chlorophylls. McGraw-Hill, New York1990: 123-161Google Scholar) in vitro. Certain other divalent metals, i.e. Mn2+, Cd2+, and Hg2+, are inhibitors(7Dailey H.A. Ann. N. Y. Acad. Sci. 1987; 514: 81-86Crossref PubMed Scopus (35) Google Scholar). Furthermore, ferric ion is not used as a substrate(8Porra R. Jones O.T.G. Biochem. J. 1963; 87: 181-185Crossref PubMed Scopus (188) Google Scholar). Deficiencies in ferrochelatase activity cause an accumulation of precursor porphyrins within cells, particularly in those tissues (i.e. liver and bone marrow) where there is a high rate of heme synthesis, and this accumulation results in the disease protoporphyria(9Nordmann Y. Deybach J.-C. Dailey H.A. Biosynthesis of Heme and Chlorophylls. McGraw-Hill, New York1990: 491-542Google Scholar). Because of this and because of heme's importance as a cofactor in a variety of enzymes and proteins (e.g. hemoglobin, cytochromes, NO synthase, peroxidases, catalases), understanding the mechanism and regulation of ferrochelatase activity is of prime importance.Ferrochelatase is a membrane-associated protein (with the cytoplasmic membrane in prokaryotes and with the inner mitochondrial membrane in eukaryotes)(2Dailey H.A. Dailey H.A. Biosynthesis of Heme and Chlorophylls. McGraw-Hill, New York1990: 123-161Google Scholar), except for the Bacillus subtilis enzyme, which is water-soluble(10Hansson M. Hederstedt L. Eur. J. Biochem. 1994; 220: 201-208Crossref PubMed Scopus (60) Google Scholar). As with most mitochondrial proteins, eukaryotic ferrochelatase is synthesized in the cytosol as a larger precursor form and subsequently processed to the mature protein during translocation into the mitochondria(2Dailey H.A. Dailey H.A. Biosynthesis of Heme and Chlorophylls. McGraw-Hill, New York1990: 123-161Google Scholar, 11Camadro J.-M. Labbe P. J. Biol. Chem. 1988; 263: 11675-11682Abstract Full Text PDF PubMed Google Scholar). Ferrochelatase genes and cDNAs have been isolated and sequenced from Escherichia coli(12Miyamoto K. Nakahigashi K. Nishimura K. Inokuchi H. J. Mol. Biol. 1991; 219: 393-398Crossref PubMed Scopus (73) Google Scholar), Bradyrhizobium japonicum(13Frustaci J. O'Brian M.R. J. Bacteriol. 1992; 174: 4223-4229Crossref PubMed Google Scholar), B. subtilis(14Hansson M. Hederstedt L. J. Bacteriol. 1992; 174: 8081-8093Crossref PubMed Google Scholar), Saccharomyces cerevisiae(15Labbe-Bois R. J. Biol. Chem. 1990; 265: 7278-7283Abstract Full Text PDF PubMed Google Scholar), Arabidopsis thaliana(16Smith A.G. Santana M.A. Wallace-Cook A.D.M. Roper J.M. Labbe-Bois R. J. Biol. Chem. 1994; 269: 13405-13413Abstract Full Text PDF PubMed Google Scholar), barley(17Miyamoto K. Tanaka R. Teramoto H. Masuda T. Tsuji H. Inokuchi H. Plant Physiol. 1994; 105: 769-770Crossref PubMed Scopus (28) Google Scholar), cucumber(17Miyamoto K. Tanaka R. Teramoto H. Masuda T. Tsuji H. Inokuchi H. Plant Physiol. 1994; 105: 769-770Crossref PubMed Scopus (28) Google Scholar), mouse (18Taketani S. Nakahashi Y. Osumi T. Tokunaga R. J. Biol. Chem. 1990; 265: 19377-19380Abstract Full Text PDF PubMed Google Scholar, 19Brenner D.A. Frasier F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 849-853Crossref PubMed Scopus (32) Google Scholar), and human(20Nakahashi Y. Taketani S. Okuda M. Inoue K. Tokunaga R. Biochem. Biophys. Res. Commun. 1990; 173: 748-755Crossref PubMed Scopus (131) Google Scholar). Human ferrochelatase is encoded by a single gene and has been mapped to chromosome 18q21.3(21Whitcombe D.M. Carter N.P. Albertson D.G. Smith S.J. Rhodes D.A. Cox T.M. Genomics. 1991; 11: 1152-1154Crossref PubMed Scopus (61) Google Scholar, 22Brenner D.A. Didier J.M. Frasier F. Christensen S.R. Evans G.A. Dailey H.A. Am. J. Hum. Genet. 1992; 50: 1203-1210PubMed Google Scholar).Because ferrochelatase is a membrane-associated protein, and hence relatively insoluble, it has been difficult in the past to purify substantial amounts of enzyme from conventional sources (e.g. mammalian liver). This has hindered detailed mechanistic and spectroscopic studies. Recently, however, this problem has been overcome by molecular recombinant DNA techniques. Both mouse (23Ferreira G.C. J. Biol. Chem. 1994; 269: 4396-4400Abstract Full Text PDF PubMed Google Scholar) and human (24Dailey H.A. Sellers V.M. Dailey T.A. J. Biol. Chem. 1994; 269: 390-395Abstract Full Text PDF PubMed Google Scholar) ferrochelatase have been overexpressed in E. coli. For the mouse enzyme, the overexpressed protein remains associated with the soluble bacterial fraction, facilitating and increasing the yields of the purification procedure(23Ferreira G.C. J. Biol. Chem. 1994; 269: 4396-4400Abstract Full Text PDF PubMed Google Scholar). Adequate amounts of the enzyme are now available, and spectroscopic studies are possible. Since these developments of heterologous overexpression systems, a [2Fe-2S] cluster was unexpectedly found in ferrochelatase isolated from mouse livers(25Ferreira B.C. Franco R. Lloyd S.G. Pereira A.S. Moura I. Moura J.J.G. Huynh B.H. J. Biol. Chem. 1994; 269: 7062-7065Abstract Full Text PDF PubMed Google Scholar), recombinant (overexpressed) mouse (25Ferreira B.C. Franco R. Lloyd S.G. Pereira A.S. Moura I. Moura J.J.G. Huynh B.H. J. Biol. Chem. 1994; 269: 7062-7065Abstract Full Text PDF PubMed Google Scholar), and recombinant human sources(26Dailey H.A. Finnegan M.G. Johnson M.K. Biochemistry. 1994; 33: 403-407Crossref PubMed Scopus (178) Google Scholar). The function of the cluster remains to be established, but it has been proposed to be necessary for activity(26Dailey H.A. Finnegan M.G. Johnson M.K. Biochemistry. 1994; 33: 403-407Crossref PubMed Scopus (178) Google Scholar). The cluster may or may not be near the ferrochelatase active site (which binds the substrate ferrous ion). The discovery of the cluster has opened new avenues of ferrochelatase research and is changing the way in which ferrochelatase in particular, and iron-sulfur clusters in general, are viewed.Little is known about the ferrous binding site itself. Chemical modification of protein sulfhydryl groups led to the proposal that at least two cysteine residues were responsible for binding the ferrous ion(27Dailey H.A. J. Biol. Chem. 1984; 259: 2711-2715Abstract Full Text PDF PubMed Google Scholar). However, comparison of the genes of the sequenced ferrochelatases reveals that not a single cysteine is conserved among all the species(12Miyamoto K. Nakahigashi K. Nishimura K. Inokuchi H. J. Mol. Biol. 1991; 219: 393-398Crossref PubMed Scopus (73) Google Scholar, 13Frustaci J. O'Brian M.R. J. Bacteriol. 1992; 174: 4223-4229Crossref PubMed Google Scholar, 14Hansson M. Hederstedt L. J. Bacteriol. 1992; 174: 8081-8093Crossref PubMed Google Scholar, 15Labbe-Bois R. J. Biol. Chem. 1990; 265: 7278-7283Abstract Full Text PDF PubMed Google Scholar, 16Smith A.G. Santana M.A. Wallace-Cook A.D.M. Roper J.M. Labbe-Bois R. J. Biol. Chem. 1994; 269: 13405-13413Abstract Full Text PDF PubMed Google Scholar, 17Miyamoto K. Tanaka R. Teramoto H. Masuda T. Tsuji H. Inokuchi H. Plant Physiol. 1994; 105: 769-770Crossref PubMed Scopus (28) Google Scholar, 18Taketani S. Nakahashi Y. Osumi T. Tokunaga R. J. Biol. Chem. 1990; 265: 19377-19380Abstract Full Text PDF PubMed Google Scholar, 19Brenner D.A. Frasier F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 849-853Crossref PubMed Scopus (32) Google Scholar, 20Nakahashi Y. Taketani S. Okuda M. Inoue K. Tokunaga R. Biochem. Biophys. Res. Commun. 1990; 173: 748-755Crossref PubMed Scopus (131) Google Scholar), although there are four cysteines conserved in the mammalian enzymes that have been implicated as ligands of the [2Fe-2S] cluster. In a recent report utilizing site-directed human ferrochelatase mutants, it was observed that the kinetic parameter KmFeincreased markedly when His-263, which is conserved in all of the sequenced ferrochelatases, was mutated to alanine while the same mutation in three other well conserved histidine residues had little effect(28Kohno H. Okuda M. Furukawa T. Tokunaga R. Taketani S. Biochim. Biophys. Acta. 1994; 1209: 95-100Crossref PubMed Scopus (48) Google Scholar). It was therefore proposed that His-263 is a ligand of the substrate iron.Identification of the residue(s) responsible for binding the substrate iron is a critical first step in elucidating the enzymatic mechanism of ferrochelatase. In this paper we report kinetic and Mössbauer data on recombinant mouse ferrochelatase, which are consistent with the proposal that histidine residues are ligands of the ferrous ion and inconsistent with the involvement of sulfur ligands. A modified purification procedure is used and an improved assay for ferrochelatase activity, which eliminates the use of the competitive iron chelator dithiothreitol (DTT),1 is described. Ferrochelatase activity was monitored by UV-visible and by Mössbauer spectroscopy, and the two techniques yielded consistent results. Mössbauer spectroscopy reveals that the ferrous heme reaction product forms the S = 0 bis(pyridine) complex upon addition of base and pyridine followed by sodium dithionite reduction and that the formation of the adduct is complete. The implications of these findings are discussed in relation to the nature of the ferrous substrate binding site.MATERIALS AND METHODSDeuteroporphyrin IX dihydrochloride and N-methylprotoporphyrin IX were purchased from Porphyrin Products (Logan, UT). Bicinchoninic acid protein assay reagents were obtained from Pierce. 57Fe metal foil (> 95% pure) was from Advanced Materials and Technology (New York, NY). Natural-abundance FeSO4 (containing 92%56Fe) was from Mallinckrodt, and natural-abundance ferrous ammonium sulfate and the sodium citrate were from Fisher. L-Ascorbic acid, ferrozine, and the ferric standard solution were from Sigma. All other chemicals were of the highest purity available.Enzyme PreparationRecombinant murine liver ferrochelatase was isolated from hyperproducing E. coli cells containing the plasmid pGF42(23Ferreira G.C. J. Biol. Chem. 1994; 269: 4396-4400Abstract Full Text PDF PubMed Google Scholar). Cells were grown in a medium containing natural-abundance iron (2.2%57Fe). The protein was purified and concentrated as described previously (23Ferreira G.C. J. Biol. Chem. 1994; 269: 4396-4400Abstract Full Text PDF PubMed Google Scholar, 25Ferreira B.C. Franco R. Lloyd S.G. Pereira A.S. Moura I. Moura J.J.G. Huynh B.H. J. Biol. Chem. 1994; 269: 7062-7065Abstract Full Text PDF PubMed Google Scholar) with the modification that the buffers used throughout the process did not contain DTT. No special anaerobic techniques were used at this stage; the protein was purified and concentrated in air. The purified, concentrated protein (typically 1-2 mM) was frozen and stored in liquid nitrogen until use. Protein concentrations were determined by the bicinchoninic acid assay using bovine serum albumin as the standard(29Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18442) Google Scholar).Ferrochelatase Activity AssayThe assays are essentially similar to those reported previously(23Ferreira G.C. J. Biol. Chem. 1994; 269: 4396-4400Abstract Full Text PDF PubMed Google Scholar), with differences noted below. Ferrochelatase was diluted into a buffer appropriate for the specific experiment (buffer and pH described for each experiment). In all cases, solutions containing ferrous ion were freshly prepared in previously deaerated distilled water (deaerated by bubbling high purity argon gas directly through).Reagents containing thiol groups, such as DTT, are well known chelators of both ferric and ferrous ion. Furthermore, thiol reagents are known to destabilize hemes in aerobic environments(30Porra R.J. Vitols K.S. Labbe R.F. Newton N.A. Biochem. J. 1967; 104: 321-327Crossref PubMed Scopus (48) Google Scholar). We therefore sought to determine if DTT, which is commonly used in the ferrochelatase activity assay, could be confounding the activity measurements. In order to assess the effects of aerobic environment and presence of reductant in the activity assays, four experiments were carried out. Four test tubes (soaked overnight in concentrated HCl to remove all traces of iron) were each filled with 0.5 ml of a 100 mM HEPES buffer solution, pH 7.5, containing 20% glycerol, 1.5 M NaCl, and 1% sodium cholate; with 0.1 ml of 2.2 μM protein solution; and with 0.1 ml of 1.88 mM deuteroporphyrin IX solution. Tubes 2 and 3 also contained 0.1 ml of 50 mM DTT solution, while tubes 1 and 4 instead contained an additional 0.1 ml of the buffer solution. All tubes were covered with a rubber stopper and placed at room temperature (22°C). Tubes 3 and 4 were deaerated for 1 h using cycles of vacuum/ultrapure argon flow. The reactions were then initiated by adding 0.05 ml of 4 mM ferrous sulfate solution. The rubber stoppers were removed from tubes 1 and 2 during the reaction, while tubes 3 and 4 remained under a flow of ultrapure, humidified argon gas. After a 30-min incubation, all tubes were opened and 0.75 ml of 1 M NaOH was added, completely stopping the reaction. The heme content was then measured using the pyridine hemochromogen method (8Porra R. Jones O.T.G. Biochem. J. 1963; 87: 181-185Crossref PubMed Scopus (188) Google Scholar) using a value of Δɛ = 15.3 mM-1 cm-1 for the reduced - oxidized difference spectra(31Porra R.J. Jones O.T.G. Biochem. J. 1963; 87: 186-192Crossref PubMed Scopus (66) Google Scholar).As we have established that DTT is not necessary for enzyme activity (see “Results”), it was not used in any subsequent activity assays. DTT is, however, a reductant that serves the role of keeping the iron in the ferrous form in aerobic assays. Therefore, without DTT, care must be taken to ensure that the assay is carried out under strictly anaerobic conditions from start to finish in order to avoid oxidation of the ferrous substrate. Therefore, the activity assay procedure was slightly modified. In a typical assay, 0.6 ml of 0.1 M Tris-HCl, pH 8.5 (or of varying pH for the pH dependence study), 0.1 ml of enzyme solution, and 0.1 ml of a 2 mM deuteroporphyrin IX solution were mixed in a stoppered test tube and deaerated for 30 min under an ultrapure argon flow. Then, 0.05 ml of a 4 mM ferrous ammonium citrate solution was added. The reaction was allowed to proceed for 20 min at 23°C and it was stopped by adding 0.5 ml of 1 M NaOH. Heme content was assayed with the pyridine hemochromogen method.To determine the optimum pH for ferrochelatase activity, activity assays were carried out using a 0.1 mM Tris-HCl buffer at pH values over the range of 6.8 to 9.7. The pH was measured at 20°C in the final reaction mixture. For the assays of ferrochelatase activity versus time of reaction, each assay contained a protein concentration of 0.105 μM.To examine the inhibition of ferrochelatase under these experimental conditions by the well studied inhibitor N-methylprotoporphyrin, stock solutions of N-methylprotoporphyrin were freshly prepared in Me2SO and then diluted into 0.1 N HCl. Each assay contained 235 μM ferrous ammonium sulfate, 235 μM deuteroporphyrin IX, and 0.4 μM ferrochelatase.Iron Determination57Fe was dissolved from metallic foil into diluted H2SO4 as described previously(32Ravi N. Bollinger J.M. Huynh B.H. Edmondson D.E. Stubbe J. J. Am. Chem. Soc. 1994; 116: 8007-8014Crossref Scopus (186) Google Scholar). After dissolution, the iron content was determined using a ferrozine colorimetric method(33Stookey L.L. Anal. Chem. 1970; 42: 779-781Crossref Scopus (3531) Google Scholar). A standard curve was made using an atomic absorption ferric standard solution. Total iron content (Fe2++ Fe3+) was determined by adding ascorbic acid to the assay mixture, while the ferrous content was determined in an assay that did not include ascorbic acid. In all cases the ferric content was small (<5%).Mössbauer SpectroscopyMössbauer spectroscopy was employed both to describe and quantitate the reaction product in an activity assay and to define the nature of the iron-binding site. To investigate the iron-binding site, a ferrous control was prepared as follows: 0.35 ml of a buffer solution (100 mM MOPS, pH 7.5, containing 10% glycerol, 1.5 M NaCl, and 1% sodium cholate) was placed in a Mössbauer sample cuvette at 23°C under argon flow and deaerated for 2 h. The lower pH is required here in order to prevent precipitation of Fe(OH)2. The sample was taken inside an anaerobic glove box (Vacuum Atmosphere Co.), and a stock solution of 57FeSO4 was added to a final concentration of 0.2 mM. The sample was then allowed to incubate for 20 min before freezing in liquid nitrogen. A protein sample was prepared in parallel. Ferrochelatase was purified and concentrated to 1.75 mM in a buffer solution identical to that of the ferrous control described above. The protein sample was placed in a Mössbauer cuvette and deaerated in a stoppered tube under ultrapure argon flow for 2 h while on ice. The sample was taken inside the glove box, and 57FeSO4 was added to a final concentration of 0.1 mM (the reason for using such a small amount of iron is discussed under “Results”). The solution was stirred and left to incubate for 20 min at 23°C. The sample was then frozen in liquid nitrogen without exposure to air.To study the interactions of ferrous ion, ferrochelatase, and DTT, a Mössbauer sample was prepared containing 1 mM57FeSO4 and 400 mM DTT in a pH 7.5 buffer as described above (sample volume is 0.35 ml). A sample was also prepared containing 310 nmol of ferrochelatase and 280 nmol of 57FeSO4; initially, this sample also contained 390 nmol of DTT. After recording the Mössbauer spectrum, a concentrated DTT stock solution was added to increase the DTT:protein ratio to 23:1 and finally to 90:1. The Mössbauer spectrum was recorded at each ratio.To quantitate and characterize the heme product, a ferrochelatase activity assay was carried out with enough material for quantitation by both the Mössbauer method and by the pyridine hemochromogen UV-visible method. The reaction mixture was prepared as follows. All reagents were deaerated as described above. In a stoppered, acid-cleaned test tube, 0.894 ml of 100 mM HEPES pH 7.5 buffer containing 20% glycerol, 1.5 M NaCl, and 1% sodium cholate was mixed with 6 μl of 74 μM ferrochelatase and 74 μl of 13.6 mM deuteroporphyrin solution. After sufficient deaeration, 26 μl of 39 mM57FeSO4 was added with a gastight syringe. The reaction was allowed to proceed under argon pressure at 24°C for 2 h, at which time 0.4 ml was transferred to a Mössbauer sample cuvette and 0.1 ml of pyridine and 0.025 ml of 4 M NaOH added. This sample was stirred and frozen in liquid nitrogen. The remainder was added to 0.75 ml of 1 M NaOH and taken for the pyridine hemochromogen assay.Mössbauer spectra were recorded on a constant-acceleration spectrometer. The instrument is equipped with a Janis 8DT variable-temperature cryostat and all measurements reported here were collected at 4.2 K. The zero velocity of the Mössbauer spectra is referenced to the centroid of the room temperature spectrum of a metallic iron foil.RESULTSDuring ferrochelatase activity determinations, we noticed that a solution of iron citrate (which is largely ferric in aerobic situations) and DTT turn a brilliant red color when mixed; this red complex slowly turns to a green color, presumably as the excess DTT reduces the ferric ion. The presence of these colored species made us suspect that DTT may be capable of binding substrate iron ions and hence interfering with the enzymatic activity. We therefore investigated this possibility with kinetic measurements and Mössbauer spectroscopy. Table 1 summarizes the effects of aerobicity and presence of DTT on ferrochelatase activity. Without DTT and in the presence of air, the enzyme activity is minimal. This is understood in terms of the oxidation state of the iron. DTT is a reductant capable of keeping iron in the ferrous state. Without it, in the presence of oxygen, the ferrous ion is expected to oxidize to the ferric form, which is not a substrate for ferrochelatase. When DTT is added to an aerobic assay, activity is observed. However, activity is increased in the anaerobic assays. Importantly, maximum activity is seen in the absence of both oxygen and DTT. The anaerobic assay that includes DTT shows only about half the activity of the assay without DTT. This difference is likely due to the ability of DTT thiol groups to competitively chelate ferrous ion and keep it from the protein. The kinetic experiments summarized here cannot directly confirm this hypothesis, but they do clearly show that DTT is not needed and that it in fact interferes with ferrochelatase activity. The lower activity seen in the aerobic assay with DTT versus the anaerobic assay with DTT possibly reflects the documented susceptibility of the heme product to attack by thiols in the presence of oxygen(30Porra R.J. Vitols K.S. Labbe R.F. Newton N.A. Biochem. J. 1967; 104: 321-327Crossref PubMed Scopus (48) Google Scholar). For these reasons we performed all subsequent measurements anaerobically and without DTT.Tabled 1 Open table in a new tab Mössbauer spectroscopy was employed to investigate the chemical environment of the substrate ferrous ion and to study the binding of ferrous ion to the enzyme. Spectra are shown in Fig. 1 and were collected at 4.2 K in the absence of an external magnetic field. Fig. 1A is the spectrum of the ferrous control in a pH 7.5 buffer. The line shapes are non-Lorentzian and cannot be fit with a single quadrupole doublet. The ferrous ion here is expected to be found in a variety of configurations and its nuclear energy levels would probably be best described with a distribution of energies. Because of this a curve fit to these data is not meaningful and therefore we report only the average values for the isomer shift d and quadrupolar splitting ΔEQ (1.39 and 3.25 mm/s, respectively). These values are consistent with high spin ferrous ions in a nitrogenous/oxygenous ligand environment (34Huynh B.H. Kent T.A. Eichhorn G.L. Marzilli L.G. Advances in Inorganic Biochemistry. 6. Elsevier Science Publishing Co., Inc., New York1984: 163-223Google Scholar) and the lack of ferric species confirms the anaerobicity of our sample preparation.Figure 1Mössbauer spectra of ferrous ion binding to ferrochelatase. A, Mössbauer spectrum of 0.2 mM57FeSO4 in buffer. The sample was prepared as described under “Materials and Methods.” The observed quadrupole doublet has parameters indicative of high spin ferrous ions in an ionic coordination environment. B, Mössbauer spectrum of 0.1 mM57FeSO4 added to 1.75 mM ferrochelatase in a buffer identical to that in spectrum A. Sample preparation is as described under “Materials and Methods.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 1B is the spectrum of 0.1 mM ferrous ion incubated with ferrochelatase. Sample preparation was similar to that of the ferrous control described above, except that the buffer solution also contained 1.75 mM ferrochelatase. The decision to use such a small amount of iron was based on the following considerations. If we assume a simple equilibrium model with three components (ferrochelatase, ferrous iron, and the enzyme-substrate complex) and define the dissociation constant for Fe(II) as KDFe=[E][Fe(II)][E-Fe(II)](Eq. 1) where [E] and [Fe(II)] are concentrations of the free ferrochelatase and unbound ferrous ion, respectively, and [E-Fe(II)] is the concentration of the complex, the ratio R of bound iron to total iron can be written as R=12{1+KDFe[Fe(II)]o+[E]o[Fe(II)]o-(1+KDFe[Fe(II)]o+[E]o[Fe(II)]o)-4[E]o[Fe(II)]o}(Eq. 2) Here [Fe(II)]o is the total concentration of iron and [E]o represents the total concentration of enzyme capable of binding iron. It may be clearly seen from that, for a given [E]o and Kd, R will be greatest for a small [Fe(II)]o. Approximating Kd with the previously determined KmFe (112 μM)(23Ferreira G.C. J. Biol. Chem. 1994; 269: 4396-4400Abstract Full Text PDF PubMed Google Scholar), which may be taken as a measure of the affinity of the active site for ferrous ion, R approaches 1.0 for [E]o≈ 2 mM and [Fe(II)]o≈ 10-1 mM. Consequently, under such conditions, the Mössbauer spectrum will represent the protein-bound Fe(II) species.Two distinct species, both quadrupole doublets, are seen in the spectrum of Fig. 1B. The first, a species with d