Studies on the Synthesis of the Fe-S Cluster of Dihydroxy-acid Dehydratase in Escherichia coli Crude Extract

The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential 4Fe-4S cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis. The inactive apoprotein could be reactivated in vitro by factors present in the crude extract of E. coli and to a much smaller extent in the presence of Fe3+, S2−, and dithiothreitol. This reactivation occurs as a result of Fe-S cluster synthesis. It is anticipated that the Fe-S cluster synthesis observed in crude extracts in vitro may involve some of the components that participate in Fe-S cluster synthesis in vivo.The origin of the sulfur used to form Fe-S clusters was investigated. Four enzymatic activities in the crude extract of E. coli were found that can provide sulfur for Fe-S cluster synthesis in vitro by mobilizing the sulfur from cysteine. The purification of the proteins responsible for three of these activities is reported in this paper. The three proteins have been identified as O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B, and β-cystathionase.The rate and extent of sulfide mobilization from cysteine in the reaction catalyzed by O-acetylserine sulfhydrylases A and B depend on the presence of nucleophiles that can add to the aminoacrylate formed on the enzyme following the removal of sulfide from cysteine. A new amino acid is formed when the nucleophiles add to the aminoacrylate. Sulfur mobilization by β-cystathionase does not require a nucleophile, and the reaction is a minor variation on the cleavage of β-cystathionine, with pyruvate, ammonia, and sulfide being the products.Once sulfur is mobilized by these enzymes, its efficient use in Fe-S cluster synthesis seems to be affected by the presence of yet unidentified factors present in crude extract. In crude extract and partially purified preparations from E. coli where these factors are present, the rapidity with which Fe-S clusters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typified by enzymatic reactions. The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential 4Fe-4S cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis. The inactive apoprotein could be reactivated in vitro by factors present in the crude extract of E. coli and to a much smaller extent in the presence of Fe3+, S2−, and dithiothreitol. This reactivation occurs as a result of Fe-S cluster synthesis. It is anticipated that the Fe-S cluster synthesis observed in crude extracts in vitro may involve some of the components that participate in Fe-S cluster synthesis in vivo. The origin of the sulfur used to form Fe-S clusters was investigated. Four enzymatic activities in the crude extract of E. coli were found that can provide sulfur for Fe-S cluster synthesis in vitro by mobilizing the sulfur from cysteine. The purification of the proteins responsible for three of these activities is reported in this paper. The three proteins have been identified as O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B, and β-cystathionase. The rate and extent of sulfide mobilization from cysteine in the reaction catalyzed by O-acetylserine sulfhydrylases A and B depend on the presence of nucleophiles that can add to the aminoacrylate formed on the enzyme following the removal of sulfide from cysteine. A new amino acid is formed when the nucleophiles add to the aminoacrylate. Sulfur mobilization by β-cystathionase does not require a nucleophile, and the reaction is a minor variation on the cleavage of β-cystathionine, with pyruvate, ammonia, and sulfide being the products. Once sulfur is mobilized by these enzymes, its efficient use in Fe-S cluster synthesis seems to be affected by the presence of yet unidentified factors present in crude extract. In crude extract and partially purified preparations from E. coli where these factors are present, the rapidity with which Fe-S clusters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typified by enzymatic reactions. INTRODUCTIONSince their discovery over 3 decades ago (Davenport et al., 8Davenport H.E. Hill R. Whatley F.R. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1952; 139: 346-358Crossref PubMed Google Scholar; Arnon et al., 1Arnon D.I. Whatley F.R. Allen M.B. Nature. 1957; 180: 182-185Crossref PubMed Scopus (38) Google Scholar; San Pietro and Lang, 53San Pietro A. Lang H.M. J. Biol. Chem. 1958; 231: 211-229Abstract Full Text PDF PubMed Google Scholar; Mortenson et al., 47Mortenson L.E. Valentine R.C. Carnahan J.E. Biochem. Biophys. Res. Commun. 1962; 7: 448-452Crossref PubMed Scopus (165) Google Scholar), proteins containing Fe-S cluster prosthetic groups have been found to play an expanding number of roles in biology. These roles now include electron transport (Johnson, 32Johnson M.K. King R.B. Encyclopedia of Inorganic Chemistry. John Wiley Lovenberg, 43Lovenberg W. Iron-Sulfur Proteins. Academic Press, New York1973Google Scholar, 44Lovenberg W. Iron-Sulfur Proteins. Academic Press, New York1973Google Scholar, 45Lovenberg W. Iron-Sulfur Proteins. Academic Press, New York1977Google Scholar; Spiro, 56Spiro T.G. Iron-Sulfur Proteins. John Wiley Emptage et al., 11Emptage M.H. Kent T.A. Kennedy M.C. Beinert H. Münck E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4674-4678Crossref PubMed Scopus (97) Google Scholar; Robbins and Stout, 51Robbins A.H. Stout C.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3639-3643Crossref PubMed Scopus (223) Google Scholar; Flint and Emptage, 14Flint D.H. Emptage M.H. J. Biol. Chem. 1988; 263: 3558-3564Abstract Full Text PDF PubMed Google Scholar; Flint et al., 16Flint D.H. Emptage M.H. Finnegan M.G. Fu W. Johnson M.K. J. Biol. Chem. 1993; 268 (a): 14732-14742Abstract Full Text PDF PubMed Google Scholar), stabilization of protein structure (Grandoni et al., 22Grandoni J.A. Switzer R.L. Makaroff C.A. Zalkin H. J. Biol. Chem. 1989; 264: 6058-6064Abstract Full Text PDF PubMed Google Scholar; Kuo et al., 37Kuo C.-F. McRee D.E. Fisher C.L. O'Handley S.F. Cunningham R.P. Rainer J.A. Science. 1992; 258: 434-440Crossref PubMed Scopus (276) Google Scholar), regulation of metabolic pathways (Bernlohr and Switzer, 4Bernlohr D.A. Switzer R.L. Biochemistry. 1981; 20: 5675-5682Crossref PubMed Scopus (25) Google Scholar; Rouault et al., 52Rouault T.A. Stout C.D. Kaptain S. Harford J.B. Klausner R.D. Cell. 1991; 64: 881-883Abstract Full Text PDF PubMed Scopus (229) Google Scholar), and a possible role in the formation of radicals (Frey and Reed, 20Frey P.A. Reed G.H. Adv. Enzymol. Relat. Areas Mol. Biol. 1993; 66: 1-39PubMed Google Scholar; Reichard, 50Reichard P. J. Biol. Chem. 1993; 268: 8383-8386Abstract Full Text PDF PubMed Google Scholar).In spite of the importance of these roles, little is known about how Fe-S clusters are made in vivo. Two general possibilities exist: spontaneous formation and enzyme-catalyzed synthesis. In support of spontaneous formation, it has been shown that protein-bound Fe-S clusters will form in vitro when the apo form of some proteins that normally contain Fe-S clusters is incubated in the presence of comparatively high concentrations of iron and sulfide (Hong and Rabinowitz, 26Hong J. Rabinowitz J.C. Biochem. Biophys. Res. Commun. 1967; 29: 246-252Crossref PubMed Scopus (54) Google Scholar; Malkin and Rabinowitz, 46Malkin R. Rabinowitz J.C. Biochem. Biophys. Res. Commun. 1966; 23: 822-827Crossref PubMed Scopus (123) Google Scholar; Yu et al., 60Yu L. Zhao J. Lu W. Bryant D.A. Golbeck J.H. Biochemistry. 1993; 32: 8251-8258Crossref PubMed Scopus (30) Google Scholar). Several of the types of Fe-S clusters that are found in proteins have been made in vitro in the absence of protein using cleverly designed organic ligands (Holm and Ibers, 25Holm R.H. Ibers J.A. Lovenberg W. Iron-Sulfur Proteins. Academic Press, New York1977: 208Google Scholar). Based on these results, some have suggested that Fe-S clusters are such thermodynamically favorable entities that formation in vivo could take place spontaneously (Lippard and Berg, 42Lippard S.J. Berg J.M. Principle of Bioinorganic Chemistry. University Science Books Mill Valley, CA1994: 115Google Scholar).In support of enzyme-catalyzed Fe-S cluster synthesis, it is known that the building blocks of Fe-S clusters, iron and sulfide, are toxic to cells at the levels required for successful Fe-S cluster synthesis in vitro (Kampfenkel et al., 33Kampfenkel K. Van Montagu M. Inze D. Plant Physiol. (Bethesda). 1995; 107: 725-735Crossref PubMed Scopus (217) Google Scholar; Gosselin and Gleason, 21Gosselin R.E. Gleason M.N. Clinical Toxicology of Commercial Products. 4th Ed. Williams PubMed Scopus Google Scholar). This could that such of enzymes for Fe-S cluster synthesis. it is possible that such enzymes to the importance of Fe-S clusters to with in these enzymes are not may be a in these enzymes that in are required a is the that Fe-S cluster formation specific enzymes to and the iron and sulfur building blocks in a controlled This to such enzymes and is an of on of the of that contain Fe-S The Fe-S clusters of these enzymes are required for enzymatic to the essential role the clusters play in catalysis (Emptage, 10Emptage M.H. Que Jr., L. Metal Clusters in Proteins. American Chemical Society, Washington, D. C.1988: 343-371Google Scholar; Flint and Emptage, 14Flint D.H. Emptage M.H. J. Biol. Chem. 1988; 263: 3558-3564Abstract Full Text PDF PubMed Google Scholar; al., D.H. Emptage M.H. Biochemistry. 1992; PubMed Scopus Google Scholar, D.H. Emptage M.H. J. Biol. Chem. 1993; 268 Full Text PDF PubMed Google to that these enzymes depend on an Fe-S cluster for could be used as in the of Fe-S cluster synthesis. Fe-S cluster synthesis on their could be even in by in enzymatic and this provide a of Fe-S cluster synthesis the formed clusters would be catalytically following the of the by using and sulfide for factors involved in sulfur for the The in et al., L. R.H. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar; and L. J. Biol. Chem. Full Text PDF PubMed Google the possible role of the protein by the from in the synthesis of the Fe-S clusters of to was a protein in Escherichia that such a protein were present in E. its role could not be formation of the Fe-S clusters of this enzyme is not found in E. such a protein have a role in the synthesis of Fe-S clusters of E. coli this were the it would that proteins are present in organisms and may have a role in Fe-S cluster synthesis. for this et al., H. D. J. 1993; PubMed Google Scholar; and D. P. J. 1993; PubMed Google and of is the that are three in the of the and in organisms have been et al., R.D. J. J.M. K. W. J. R. L. A. J.M. C.A. E. M.C. R.C. C.L. J.C. Science. 1995; PubMed Scopus Google Scholar; et al., R.D. J.M. M. J. D. C.A. J.C. Science. 1995; PubMed Scopus Google reaction catalyzed by in the presence of is the formation of sulfide and from cysteine as shown in where and to and a reaction place in E. coli crude it was and found to have the to sulfide from cysteine in the presence of dithiothreitol. such as enzymes that will sulfur from cysteine for use in the formation of Fe-S clusters have been purified and identified from the crude extract of E. The purification and of the three of these enzymes are in this and the purification of the enzyme is in the following D.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google this have reported on Fe-S cluster synthesis. The are the inactive apo form of E. coli dihydroxy-acid in vivo by cells with in vitro by the enzyme with a of and can be reactivated in vitro by factors present in E. coli crude extract. are at least enzymes in the crude extract of E. coli that can the sulfur from cysteine and to the synthesis of Fe-S clusters in vitro in crude extract. the efficiency of use of the sulfur mobilized from cysteine in Fe-S cluster synthesis is high in the from the can be reactivated to a extent in the presence of Fe3+, S2−, and dithiothreitol. The rate and extent of reactivation in this are low with place in the presence of factors from the crude extract. the efficiency of sulfur in the presence of factors from the crude extract an orderly controlled formation of Fe-S clusters that is typified generally by enzymatic reactions. This is in to a formation of Fe-S clusters in the presence of Fe3+, S2−, and which to be on the O-acetylserine sulfhydrylase A and mobilization of sulfur from cysteine has been shown to be to a to the aminoacrylate by the of a a of nucleophiles for their to add to the aminoacrylate formed on O-acetylserine sulfhydrylase A. The at which the nucleophiles add not with their in reactions. the of and sulfide in reactions is J. Chemistry. Ed. Co., New Scholar), and add very at to the aminoacrylate formed on O-acetylserine sulfhydrylase A, at an and sulfide This in be by not the to the active of the enzyme by the of the The of the the enzyme the of of these nucleophiles are of importance in its mobilization of sulfur from cysteine is not to be the reaction catalyzed by O-acetylserine sulfhydrylases A and B and β-cystathionase. with this is that these enzymes catalyze the mobilization of sulfur from cysteine at a much rate catalyze the reactions to be their This up the of these three enzymes participate in the synthesis of Fe-S clusters in is that these enzymes are not involved in Fe-S cluster synthesis in vivo, the in vitro is a of the reaction can catalyze the in this the reactions have found to in vivo in the of O-acetylserine sulfhydrylases A and B, nucleophiles such as would have to play the role in This would to the formation of amino that as as not exist in vivo. does not such a to sulfur from its in with would a in organisms that cysteine in which sulfide was and from the a of enzymes does exist to the formation of Fe-S clusters by a would that the of enzymes and the would be the the enzymes in this in the organisms that not exist in organisms such as that cysteine. It seems that a of enzymes does exist to the formation of Fe-S clusters, the sulfur would be by in such a that its could be controlled Fe-S cluster formation, which does not to be the for the following for a of a in which the of the sulfur could be controlled D.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have an and designed an that can be used to the of with nucleophiles that add to the aminoacrylate formed on O-acetylserine sulfhydrylases A and This and may be of in the active of these enzymes and in are some minor the of this and reported by et D.E. Biochemistry. 1993; 32: PubMed Scopus Google the of on O-acetylserine sulfhydrylases A and These reported that in the form not the reaction catalyzed by the S. this is not of E. coli O-acetylserine sulfhydrylases A and B these enzymes can use as a the S. O-acetylserine sulfhydrylases can use acid as a substrate et al., D.E. Biochemistry. 1993; 32: PubMed Scopus Google Scholar), it would be could not use have shown that O-acetylserine sulfhydrylase A can form from this place by of the of to the aminoacrylate formed from This up a of et S. S. Biochemistry. 1992; PubMed Scopus Google have to the of O-acetylserine sulfhydrylase in which O-acetylserine is to pyruvate, ammonia, and as a The would to be to the of O-acetylserine to and that have and the et to as a be to as a reactions much to a and the aminoacrylate to by the of to of the aminoacrylate and the by the of to the to the aminoacrylate to the free which to and be that this is the to that O-acetylserine sulfhydrylases can a of nucleophiles sulfide for to the aminoacrylate formed from this of reaction has been in the of O-acetylserine sulfhydrylases from et al., M. M. 1988; Scholar, M. M. Scopus Google Scholar, S. 1993; 32: Scopus Google Scholar). to have for the the role of nucleophiles in the from cysteine. of the nucleophiles reported to be a substrate for enzymes is the et al., M. (a): Scopus Google Scholar). It has been that the formation of the of the of to the aminoacrylate reported in this may be responsible for the known of and A. Res. 1988; PubMed Scopus Google important to the reported in this is its in Fe-S cluster synthesis in vivo. some of the factors and reactions that place in the crude extracts used in this to Fe-S cluster synthesis are involved in the synthesis of Fe-S clusters in vivo, this is of The and of the apoprotein of dihydroxy-acid to the in the presence of E. coli crude extract to for how Fe-S clusters are made in vivo. It is that is a cluster and of formation, the Fe-S clusters formed on the used in these have been formed the Fe-S cluster of this enzyme is et al., 16Flint D.H. Emptage M.H. Finnegan M.G. Fu W. Johnson M.K. J. Biol. Chem. 1993; 268 (a): 14732-14742Abstract Full Text PDF PubMed Google Scholar). INTRODUCTIONSince their discovery over 3 decades ago (Davenport et al., 8Davenport H.E. Hill R. Whatley F.R. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1952; 139: 346-358Crossref PubMed Google Scholar; Arnon et al., 1Arnon D.I. Whatley F.R. Allen M.B. Nature. 1957; 180: 182-185Crossref PubMed Scopus (38) Google Scholar; San Pietro and Lang, 53San Pietro A. Lang H.M. J. Biol. Chem. 1958; 231: 211-229Abstract Full Text PDF PubMed Google Scholar; Mortenson et al., 47Mortenson L.E. Valentine R.C. Carnahan J.E. Biochem. Biophys. Res. Commun. 1962; 7: 448-452Crossref PubMed Scopus (165) Google Scholar), proteins containing Fe-S cluster prosthetic groups have been found to play an expanding number of roles in biology. These roles now include electron transport (Johnson, 32Johnson M.K. King R.B. Encyclopedia of Inorganic Chemistry. John Wiley Lovenberg, 43Lovenberg W. Iron-Sulfur Proteins. Academic Press, New York1973Google Scholar, 44Lovenberg W. Iron-Sulfur Proteins. Academic Press, New York1973Google Scholar, 45Lovenberg W. Iron-Sulfur Proteins. Academic Press, New York1977Google Scholar; Spiro, 56Spiro T.G. Iron-Sulfur Proteins. John Wiley Emptage et al., 11Emptage M.H. Kent T.A. Kennedy M.C. Beinert H. Münck E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4674-4678Crossref PubMed Scopus (97) Google Scholar; Robbins and Stout, 51Robbins A.H. Stout C.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3639-3643Crossref PubMed Scopus (223) Google Scholar; Flint and Emptage, 14Flint D.H. Emptage M.H. J. Biol. Chem. 1988; 263: 3558-3564Abstract Full Text PDF PubMed Google Scholar; Flint et al., 16Flint D.H. Emptage M.H. Finnegan M.G. Fu W. Johnson M.K. J. Biol. Chem. 1993; 268 (a): 14732-14742Abstract Full Text PDF PubMed Google Scholar), stabilization of protein structure (Grandoni et al., 22Grandoni J.A. Switzer R.L. Makaroff C.A. Zalkin H. J. Biol. Chem. 1989; 264: 6058-6064Abstract Full Text PDF PubMed Google Scholar; Kuo et al., 37Kuo C.-F. McRee D.E. Fisher C.L. O'Handley S.F. Cunningham R.P. Rainer J.A. Science. 1992; 258: 434-440Crossref PubMed Scopus (276) Google Scholar), regulation of metabolic pathways (Bernlohr and Switzer, 4Bernlohr D.A. Switzer R.L. Biochemistry. 1981; 20: 5675-5682Crossref PubMed Scopus (25) Google Scholar; Rouault et al., 52Rouault T.A. Stout C.D. Kaptain S. Harford J.B. Klausner R.D. Cell. 1991; 64: 881-883Abstract Full Text PDF PubMed Scopus (229) Google Scholar), and a possible role in the formation of radicals (Frey and Reed, 20Frey P.A. Reed G.H. Adv. Enzymol. Relat. Areas Mol. Biol. 1993; 66: 1-39PubMed Google Scholar; Reichard, 50Reichard P. J. Biol. Chem. 1993; 268: 8383-8386Abstract Full Text PDF PubMed Google Scholar).In spite of the importance of these roles, little is known about how Fe-S clusters are made in vivo. Two general possibilities exist: spontaneous formation and enzyme-catalyzed synthesis. In support of spontaneous formation, it has been shown that protein-bound Fe-S clusters will form in vitro when the apo form of some proteins that normally contain Fe-S clusters is incubated in the presence of comparatively high concentrations of iron and sulfide (Hong and Rabinowitz, 26Hong J. Rabinowitz J.C. Biochem. Biophys. Res. Commun. 1967; 29: 246-252Crossref PubMed Scopus (54) Google Scholar; Malkin and Rabinowitz, 46Malkin R. Rabinowitz J.C. Biochem. Biophys. Res. Commun. 1966; 23: 822-827Crossref PubMed Scopus (123) Google Scholar; Yu et al., 60Yu L. Zhao J. Lu W. Bryant D.A. Golbeck J.H. Biochemistry. 1993; 32: 8251-8258Crossref PubMed Scopus (30) Google Scholar). Several of the types of Fe-S clusters that are found in proteins have been made in vitro in the absence of protein using cleverly designed organic ligands (Holm and Ibers, 25Holm R.H. Ibers J.A. Lovenberg W. Iron-Sulfur Proteins. Academic Press, New York1977: 208Google Scholar). Based on these results, some have suggested that Fe-S clusters are such thermodynamically favorable entities that formation in vivo could take place spontaneously (Lippard and Berg, 42Lippard S.J. Berg J.M. Principle of Bioinorganic Chemistry. University Science Books Mill Valley, CA1994: 115Google Scholar).In support of enzyme-catalyzed Fe-S cluster synthesis, it is known that the building blocks of Fe-S clusters, iron and sulfide, are toxic to cells at the levels required for successful Fe-S cluster synthesis in vitro (Kampfenkel et al., 33Kampfenkel K. Van Montagu M. Inze D. Plant Physiol. (Bethesda). 1995; 107: 725-735Crossref PubMed Scopus (217) Google Scholar; Gosselin and Gleason, 21Gosselin R.E. Gleason M.N. Clinical Toxicology of Commercial Products. 4th Ed. Williams PubMed Scopus Google Scholar). This could that such of enzymes for Fe-S cluster synthesis. it is possible that such enzymes to the importance of Fe-S clusters to with in these enzymes are not may be a in these enzymes that in are required a is the that Fe-S cluster formation specific enzymes to and the iron and sulfur building blocks in a controlled This to such enzymes and is an of on of the of that contain Fe-S The Fe-S clusters of these enzymes are required for enzymatic to the essential role the clusters play in catalysis (Emptage, 10Emptage M.H. Que Jr., L. Metal Clusters in Proteins. American Chemical Society, Washington, D. C.1988: 343-371Google Scholar; Flint and Emptage, 14Flint D.H. Emptage M.H. J. Biol. Chem. 1988; 263: 3558-3564Abstract Full Text PDF PubMed Google Scholar; al., D.H. Emptage M.H. Biochemistry. 1992; PubMed Scopus Google Scholar, D.H. Emptage M.H. J. Biol. Chem. 1993; 268 Full Text PDF PubMed Google to that these enzymes depend on an Fe-S cluster for could be used as in the of Fe-S cluster synthesis. Fe-S cluster synthesis on their could be even in by in enzymatic and this provide a of Fe-S cluster synthesis the formed clusters would be catalytically following the of the by using and sulfide for factors involved in sulfur for the The in et al., L. R.H. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar; and L. J. Biol. Chem. Full Text PDF PubMed Google the possible role of the protein by the from in the synthesis of the Fe-S clusters of to was a protein in Escherichia that such a protein were present in E. its role could not be formation of the Fe-S clusters of this enzyme is not found in E. such a protein have a role in the synthesis of Fe-S clusters of E. coli this were the it would that proteins are present in organisms and may have a role in Fe-S cluster synthesis. for this et al., H. D. J. 1993; PubMed Google Scholar; and D. P. J. 1993; PubMed Google and of is the that are three in the of the and in organisms have been et al., R.D. J. J.M. K. W. J. R. L. A. J.M. C.A. E. M.C. R.C. C.L. J.C. Science. 1995; PubMed Scopus Google Scholar; et al., R.D. J.M. M. J. D. C.A. J.C. Science. 1995; PubMed Scopus Google reaction catalyzed by in the presence of is the formation of sulfide and from cysteine as shown in where and to and a reaction place in E. coli crude it was and found to have the to sulfide from cysteine in the presence of dithiothreitol. such as enzymes that will sulfur from cysteine for use in the formation of Fe-S clusters have been purified and identified from the crude extract of E. The purification and of the three of these enzymes are in this and the purification of the enzyme is in the following D.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar).