Abstract
In bacteria, HscB is the cochaperone of HscA in modulating the transfer of 2Fe2S clusters from a cluster-loaded form of the scaffold protein IscU to acceptor apoproteins. HscB binding to the IscU apoform (apoIscU) reportedly impairs the structural flexibility of apoIscU, but the effects of HscB on cluster formation on IscU have never been assessed. We report that presence of HscB impaired the rate—but not the equilibrium—of the appearance of the distinctive circular dichroism signals associated with formation of a stable 2Fe–2S cluster on IscU in reconstitution experiments. This impairment: (1) was independent of the source of cluster sulfide; (2) was not observed for HscB mutants unable to bind IscU; (3) implied formation of a 1/1 HscB/IscU complex; (4) was not observed for a D39A mutant of IscU, with a much more rigid structure than wt IscU. The cluster species assembled on IscU in the presence of HscB were transferred to apoferredoxin at a slower rate than those formed in the absence of HscB, unless ATP and HscA were also present. At contrast, HscB was found to improve the “catalytic” function of IscU with respect to cluster assembly in the presence of a large apoferredoxin excess. Thus, the HscB/IscU interaction may modulate formation and transfer of FeS clusters by accelerating cluster biosynthesis when appropriate target apoproteins are abundant or by slowing it down when the rate of apoprotein synthesis is slow, and cluster-loaded IscU is more likely to play a role as a “FeS storage” protein.
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Abbreviations
- CD:
-
Circular dichroism
- TD buffer:
-
0.1 M Tris–HCl, pH 8.0, 5 mM dithiothreitol
- TDMK buffer:
-
0.1 M Tris–HCl, pH 8.0, 5 mM dithiothreitol, 10 mM MgCl2, 150 mM KCl
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Acknowledgments
The authors would like to express their gratitude to Dr. Dennis D. Ta and to Prof. Larry E. Vickery, formerly at UC-Irvine, for providing most of the proteins used in this study.
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Iametti, S., Barbiroli, A. & Bonomi, F. Functional implications of the interaction between HscB and IscU in the biosynthesis of FeS clusters. J Biol Inorg Chem 20, 1039–1048 (2015). https://doi.org/10.1007/s00775-015-1285-z
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DOI: https://doi.org/10.1007/s00775-015-1285-z