Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide

Robert M. Cicchillo, Kyung Hoon Lee, Camelia Baleanu-Gogonea, Natasha M. Nesbitt, Carsten Krebs, Squire J. Booker

Research output: Contribution to journalArticle

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Abstract

Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-L-methionine (SAM) as the precursor to a high-energy 5′-deoxyadenosyl 5′-radical (5′-dA.). In the LS reaction, the 5′-dA. is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d15 H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X3-C-X2-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X4-C-X5-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 ± 0.5 irons and 6.4 ± 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5′-deoxyadenosine (5′-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 ± 0.1 irons and 3.6 ± 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 ± 0.1 irons and 4.7 ± 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5′-dA or the lipoyl group. Mössbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S]2+. When wild-type LS was reconstituted with 57Fe and sodium sulfide, it harbored considerably more iron (13.8 ± 0.6) and sulfide (13.1 ± 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5′-dA and 0.36 equiv of the lipoyl group. Mössbauer spectroscopy of this protein revealed that only ∼67% ± 6% of the iron is in the form of [4Fe-4S]2+ clusters, amounting to 9.2 ± 0.4 irons and 8.8 ± 0.1 sulfides or 2 [4Fe-4S]2+ clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the Mössbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.

Original languageEnglish (US)
Pages (from-to)11770-11781
Number of pages12
JournalBiochemistry
Volume43
Issue number37
DOIs
StatePublished - Sep 21 2004

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Escherichia coli
Sulfides
Peptides
S-Adenosylmethionine
Iron
Cysteine
Proteins
Glycine Decarboxylase Complex H-Protein
Sulfur
Spectrum Analysis
Enzymes
Spectroscopy
Azotobacter vinelandii
Amino Acids
Biochemistry
Deuterium
Biosynthesis
Operon
Ports and harbors
Protein C

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Cicchillo, Robert M. ; Lee, Kyung Hoon ; Baleanu-Gogonea, Camelia ; Nesbitt, Natasha M. ; Krebs, Carsten ; Booker, Squire J. / Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide. In: Biochemistry. 2004 ; Vol. 43, No. 37. pp. 11770-11781.
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title = "Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide",
abstract = "Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-L-methionine (SAM) as the precursor to a high-energy 5′-deoxyadenosyl 5′-radical (5′-dA.). In the LS reaction, the 5′-dA. is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d15 H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X3-C-X2-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X4-C-X5-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 ± 0.5 irons and 6.4 ± 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5′-deoxyadenosine (5′-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 ± 0.1 irons and 3.6 ± 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 ± 0.1 irons and 4.7 ± 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5′-dA or the lipoyl group. M{\"o}ssbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90{\%} of all associated iron is in the configuration [4Fe-4S]2+. When wild-type LS was reconstituted with 57Fe and sodium sulfide, it harbored considerably more iron (13.8 ± 0.6) and sulfide (13.1 ± 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5′-dA and 0.36 equiv of the lipoyl group. M{\"o}ssbauer spectroscopy of this protein revealed that only ∼67{\%} ± 6{\%} of the iron is in the form of [4Fe-4S]2+ clusters, amounting to 9.2 ± 0.4 irons and 8.8 ± 0.1 sulfides or 2 [4Fe-4S]2+ clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the M{\"o}ssbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.",
author = "Cicchillo, {Robert M.} and Lee, {Kyung Hoon} and Camelia Baleanu-Gogonea and Nesbitt, {Natasha M.} and Carsten Krebs and Booker, {Squire J.}",
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Cicchillo, RM, Lee, KH, Baleanu-Gogonea, C, Nesbitt, NM, Krebs, C & Booker, SJ 2004, 'Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide', Biochemistry, vol. 43, no. 37, pp. 11770-11781. https://doi.org/10.1021/bi0488505

Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide. / Cicchillo, Robert M.; Lee, Kyung Hoon; Baleanu-Gogonea, Camelia; Nesbitt, Natasha M.; Krebs, Carsten; Booker, Squire J.

In: Biochemistry, Vol. 43, No. 37, 21.09.2004, p. 11770-11781.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide

AU - Cicchillo, Robert M.

AU - Lee, Kyung Hoon

AU - Baleanu-Gogonea, Camelia

AU - Nesbitt, Natasha M.

AU - Krebs, Carsten

AU - Booker, Squire J.

PY - 2004/9/21

Y1 - 2004/9/21

N2 - Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-L-methionine (SAM) as the precursor to a high-energy 5′-deoxyadenosyl 5′-radical (5′-dA.). In the LS reaction, the 5′-dA. is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d15 H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X3-C-X2-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X4-C-X5-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 ± 0.5 irons and 6.4 ± 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5′-deoxyadenosine (5′-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 ± 0.1 irons and 3.6 ± 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 ± 0.1 irons and 4.7 ± 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5′-dA or the lipoyl group. Mössbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S]2+. When wild-type LS was reconstituted with 57Fe and sodium sulfide, it harbored considerably more iron (13.8 ± 0.6) and sulfide (13.1 ± 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5′-dA and 0.36 equiv of the lipoyl group. Mössbauer spectroscopy of this protein revealed that only ∼67% ± 6% of the iron is in the form of [4Fe-4S]2+ clusters, amounting to 9.2 ± 0.4 irons and 8.8 ± 0.1 sulfides or 2 [4Fe-4S]2+ clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the Mössbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.

AB - Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-L-methionine (SAM) as the precursor to a high-energy 5′-deoxyadenosyl 5′-radical (5′-dA.). In the LS reaction, the 5′-dA. is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d15 H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X3-C-X2-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X4-C-X5-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 ± 0.5 irons and 6.4 ± 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5′-deoxyadenosine (5′-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 ± 0.1 irons and 3.6 ± 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 ± 0.1 irons and 4.7 ± 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5′-dA or the lipoyl group. Mössbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S]2+. When wild-type LS was reconstituted with 57Fe and sodium sulfide, it harbored considerably more iron (13.8 ± 0.6) and sulfide (13.1 ± 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5′-dA and 0.36 equiv of the lipoyl group. Mössbauer spectroscopy of this protein revealed that only ∼67% ± 6% of the iron is in the form of [4Fe-4S]2+ clusters, amounting to 9.2 ± 0.4 irons and 8.8 ± 0.1 sulfides or 2 [4Fe-4S]2+ clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the Mössbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.

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