Iron-sulfur cluster biosynthesis. A comparative kinetic analysis of native and Cys-substituted ISA-mediated [2Fe-2S]2+ cluster transfer to an apoferredoxin target

Shu-Pao Wu, J. A. Cowan*

*Corresponding author for this work

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

ISA type proteins mediate cluster transfer to apoprotein targets. Rate constants have been determined for cluster transfer from Schizosaccharomyces pombe ISA to apo Fd. Substitution of the cysteine residues of ISA produced derivative proteins (C72A, C136A, and C138A) that were found to be at least as active in cluster transfer reactions as the native form at 25°C (k2 ∼ 170 M-1 min-1 for native, k2 ∼ 169 M-1 min-1 for C72A, k2 ∼ 206 M-1 min-1 for C136A, and k2 ∼ 242 M-1 min-1 for C138A), although the yield of cluster transfer was found to be lower as a consequence of the enhanced lability of clusters in the derivative proteins. Minor variations in rate constant for the ISA Cys derivatives do not reflect any change in the affinity of binding to the apo Fd since k2 was found to be independent of the concentration of apo Fd over the range of 1-25 μM. The pH dependence of cluster transfer rates was found to be similar for native and C136A ISA, with an observed pKa of 7.8 determined from the pH profiles for cluster transfer activity of each protein. The temperature dependence of the rate constant defining the cluster transfer reaction for the wild type versus this C136A ISA derivative is distinct (ΔH* ∼ 6.3 kcal mol-1 and ΔS* ∼ -27.3 cal K-1 mol-1 for native and ΔH* ∼ 2.7 kcal mol-1 and ΔS* ∼ -38.9 cal K-1 mol-1 for C136A ISA). Instability of the protein-bound cluster precluded a comparison with data from pH and temperature dependencies for the two other Cys derivatives. Experiments to determine the dependence of reaction rate constants on viscosity indicate cluster transfer is rate-limiting. A comparison of cross-species rate constants for cluster transfer to apo Fd targets from Homo sapiens and S. pombe demonstrated that the identity of the Fd is less critical for promoting cluster transfer from Sp ISA (at 25°C, k2 ∼ 170 M-1 min-1 for Sp Fd and k2 ∼ 169 M-1 min-1 for Hs Fd). This contrasts with an earlier observation for ISU-mediated cluster assembly [Wu, S., et al. (2002) Biochemistry 41, 8876-8885], where the rates differed for Hs and Sp target Fd's, suggesting distinct binding sites for binding of holo ISA and ISU to apo Fd.

Original languageEnglish
Pages (from-to)5784-5791
Number of pages8
JournalBiochemistry
Volume42
Issue number19
DOIs
StatePublished - 20 May 2003

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