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Characterization of an iron-sulfur cluster assembly protein (ISU1) from Schizosaccharomyces pombe.

Publication ,  Journal Article
Wu, G; Mansy, SS; Wu Sp, S-P; Surerus, KK; Foster, MW; Cowan, JA
Published in: Biochemistry
April 16, 2002

Genetic studies of bacteria and eukaryotes have led to identification of several gene products that are involved in the biosynthesis of protein-bound iron-sulfur clusters. One of these proteins, ISU, is homologous to the N-terminus of bacterial NifU. The mature forms of His-tagged wild-type and D37A Schizosaccharomyces pombe ISU1 were cloned and overexpressed as inclusion bodies in Escherichia coli. The recombinant D37A protein was purified under denaturing conditions and subsequently reconstituted in vitro. By use of a 5-fold excess of iron and sulfide the reconstituted product was found to be red-brown in color, forming a homodimer of 17 kDa per subunit with approximately two iron atoms per monomer determined by protein and iron quantitation. UV-vis absorption and Mössbauer spectroscopies (delta = 0.29 +/- 0.05 mm/s; DeltaE(Q) = 0.59 +/- 0.05 mm/s) were used to characterize D37A ISU1 and show the presence of [2Fe-2S](2+) clusters in each subunit. Formation of the holo form of wild-type ISU1 was significantly less efficient using the same reconstitution conditions and is consistent with prior observations that the D37A substitution can stabilize protein-bound clusters. Relative to the human homologue, the yeast ISU is significantly less soluble at ambient temperatures. In both cases the native ISU1 is more sensitive to proton-mediated degradation relative to the D37A derivative. The lability of this family of proteins relative to [2Fe-2S] bearing ferredoxins most likely is of functional relevance for cluster transfer chemistry. Mössbauer parameters obtained for wild-type ISU1 (delta = 0.31 +/- 0.05 mm/s; DeltaE(Q) = 0.64 +/- 0.05 mm/s) were similar to those obtained for the D37A derivative. Cluster transfer from ISU1 to apo Fd is demonstrated: the first example of transfer from an ISU-type protein. A lower limit for k(2) of 80 M(-1) min(-1) was established for WT cluster transfer and a value of 18 M(-1) min(-1) for the D37A derivative. Finally, we have demonstrated through cross-linking studies that ferredoxin, an electron-transport protein, forms a complex with ISU1 in both apo and holo states. Cross-linking of holo ISU1 with holo Fd is consistent with a role for redox chemistry in cluster assembly and may mimic the intramolecular complex already defined in NifU.

Duke Scholars

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

April 16, 2002

Volume

41

Issue

15

Start / End Page

5024 / 5032

Location

United States

Related Subject Headings

  • Sequence Homology, Amino Acid
  • Sequence Alignment
  • Schizosaccharomyces
  • Saccharomyces cerevisiae Proteins
  • Recombinant Proteins
  • Protein Subunits
  • Protein Denaturation
  • Polymerase Chain Reaction
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
 

Citation

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MLA
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Wu, G., Mansy, S. S., Wu Sp, S.-P., Surerus, K. K., Foster, M. W., & Cowan, J. A. (2002). Characterization of an iron-sulfur cluster assembly protein (ISU1) from Schizosaccharomyces pombe. Biochemistry, 41(15), 5024–5032. https://doi.org/10.1021/bi016073s
Wu, Gong, Sheref S. Mansy, Shu-pao Wu Sp, Kristene K. Surerus, Matthew W. Foster, and J. A. Cowan. “Characterization of an iron-sulfur cluster assembly protein (ISU1) from Schizosaccharomyces pombe.Biochemistry 41, no. 15 (April 16, 2002): 5024–32. https://doi.org/10.1021/bi016073s.
Wu G, Mansy SS, Wu Sp S-P, Surerus KK, Foster MW, Cowan JA. Characterization of an iron-sulfur cluster assembly protein (ISU1) from Schizosaccharomyces pombe. Biochemistry. 2002 Apr 16;41(15):5024–32.
Wu, Gong, et al. “Characterization of an iron-sulfur cluster assembly protein (ISU1) from Schizosaccharomyces pombe.Biochemistry, vol. 41, no. 15, Apr. 2002, pp. 5024–32. Pubmed, doi:10.1021/bi016073s.
Wu G, Mansy SS, Wu Sp S-P, Surerus KK, Foster MW, Cowan JA. Characterization of an iron-sulfur cluster assembly protein (ISU1) from Schizosaccharomyces pombe. Biochemistry. 2002 Apr 16;41(15):5024–5032.

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

April 16, 2002

Volume

41

Issue

15

Start / End Page

5024 / 5032

Location

United States

Related Subject Headings

  • Sequence Homology, Amino Acid
  • Sequence Alignment
  • Schizosaccharomyces
  • Saccharomyces cerevisiae Proteins
  • Recombinant Proteins
  • Protein Subunits
  • Protein Denaturation
  • Polymerase Chain Reaction
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data