IscA, an alternate scaffold for Fe-S cluster biosynthesis

Carsten Krebs, J. N. Agar, A. D. Smith, J. Frazzon, D. R. Dean, B. H. Huynh, M. K. Johnson

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Abstract

An IscA homologue within the nif regulon of Azotobacter vinelandii, designated NifIscA, was expressed in Escherichia coli and purified to homogeneity. Purified NifIscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for NifIscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mössbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although NifIscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for NifIscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S]2+ cluster per NifIscA homodimer, via a transient [2Fe-2S]2+ cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and M6ssbauer spectroscopy, and the intermediate [2Fe-2S]2+-containing species was additionally characterized by resonance Raman spectroscopy. The Mössbauer and resonance Raman properties of the [2Fe-2S]2+ center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S]2+ or one [4Fe-4S]2+ per homodimer suggest that both cluster types are subunit bridging. In addition, NifIscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on NifIscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.

Original languageEnglish (US)
Pages (from-to)14069-14080
Number of pages12
JournalBiochemistry
Volume40
Issue number46
DOIs
StatePublished - Nov 20 2001

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Biosynthesis
Scaffolds
Ions
Protein S
Sulfur
Metallochaperones
Spectrum Analysis
Proteins
Iron
Azotobacter vinelandii
Regulon
Raman Spectrum Analysis
Poisons
Circular Dichroism
Spectroscopy
Cysteine
Ligation
Metals
Escherichia coli
Prosthetics

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Krebs, C., Agar, J. N., Smith, A. D., Frazzon, J., Dean, D. R., Huynh, B. H., & Johnson, M. K. (2001). IscA, an alternate scaffold for Fe-S cluster biosynthesis. Biochemistry, 40(46), 14069-14080. https://doi.org/10.1021/bi015656z
Krebs, Carsten ; Agar, J. N. ; Smith, A. D. ; Frazzon, J. ; Dean, D. R. ; Huynh, B. H. ; Johnson, M. K. / IscA, an alternate scaffold for Fe-S cluster biosynthesis. In: Biochemistry. 2001 ; Vol. 40, No. 46. pp. 14069-14080.
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Krebs, C, Agar, JN, Smith, AD, Frazzon, J, Dean, DR, Huynh, BH & Johnson, MK 2001, 'IscA, an alternate scaffold for Fe-S cluster biosynthesis', Biochemistry, vol. 40, no. 46, pp. 14069-14080. https://doi.org/10.1021/bi015656z

IscA, an alternate scaffold for Fe-S cluster biosynthesis. / Krebs, Carsten; Agar, J. N.; Smith, A. D.; Frazzon, J.; Dean, D. R.; Huynh, B. H.; Johnson, M. K.

In: Biochemistry, Vol. 40, No. 46, 20.11.2001, p. 14069-14080.

Research output: Contribution to journalArticle

TY - JOUR

T1 - IscA, an alternate scaffold for Fe-S cluster biosynthesis

AU - Krebs, Carsten

AU - Agar, J. N.

AU - Smith, A. D.

AU - Frazzon, J.

AU - Dean, D. R.

AU - Huynh, B. H.

AU - Johnson, M. K.

PY - 2001/11/20

Y1 - 2001/11/20

N2 - An IscA homologue within the nif regulon of Azotobacter vinelandii, designated NifIscA, was expressed in Escherichia coli and purified to homogeneity. Purified NifIscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for NifIscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mössbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although NifIscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for NifIscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S]2+ cluster per NifIscA homodimer, via a transient [2Fe-2S]2+ cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and M6ssbauer spectroscopy, and the intermediate [2Fe-2S]2+-containing species was additionally characterized by resonance Raman spectroscopy. The Mössbauer and resonance Raman properties of the [2Fe-2S]2+ center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S]2+ or one [4Fe-4S]2+ per homodimer suggest that both cluster types are subunit bridging. In addition, NifIscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on NifIscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.

AB - An IscA homologue within the nif regulon of Azotobacter vinelandii, designated NifIscA, was expressed in Escherichia coli and purified to homogeneity. Purified NifIscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for NifIscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mössbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although NifIscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for NifIscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S]2+ cluster per NifIscA homodimer, via a transient [2Fe-2S]2+ cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and M6ssbauer spectroscopy, and the intermediate [2Fe-2S]2+-containing species was additionally characterized by resonance Raman spectroscopy. The Mössbauer and resonance Raman properties of the [2Fe-2S]2+ center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S]2+ or one [4Fe-4S]2+ per homodimer suggest that both cluster types are subunit bridging. In addition, NifIscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on NifIscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.

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Krebs C, Agar JN, Smith AD, Frazzon J, Dean DR, Huynh BH et al. IscA, an alternate scaffold for Fe-S cluster biosynthesis. Biochemistry. 2001 Nov 20;40(46):14069-14080. https://doi.org/10.1021/bi015656z