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Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly

Urbina, Hugo D. and Silberg, Jonathan J. and Hoff, Kevin G. and Vickery, Larry E. (2001) Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly. Journal of Biological Chemistry, 276 (48). pp. 44521-44526. ISSN 0021-9258. https://resolver.caltech.edu/CaltechAUTHORS:URBjbc01

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Abstract

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [35S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of 35S from [35S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (Kd ~2 µM) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and 35S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta 376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer 35S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta 376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.


Item Type:Article
Additional Information:Copyright © 2001 by the American Society for Biochemistry and Molecular Biology. Received for publication, July 20, 2001, and in revised form, September 26, 2001. Originally published In Press as doi:10.1074/jbc.M106907200 on September 27, 2001 We thank Dr. Patricia Kiley for providing iscS disruption strain PK4331. This work was supported by National Institutes of Health Grant GM54264 and Training Grant GM07311.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Issue or Number:48
Record Number:CaltechAUTHORS:URBjbc01
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:URBjbc01
Alternative URL:http://dx.doi.org/10.1074/jbc.M106907200
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5838
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:04 Nov 2006
Last Modified:02 Oct 2019 23:27

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