Transfer of sulfur from IscS to IscU during Fe/S cluster assembly.
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 [(35)S]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 (35)S from [(35)S]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 (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S 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 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 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.
Urbina
H D
HD
Department of Physiology, University of California, Irvine, California 92697, USA.
Silberg
J J
JJ
Hoff
K G
KG
Vickery
L E
LE
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D013437:Carbon-Sulfur Lyases / Q000378:metabolism*; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C411665:IscU protein, E coli; D011994:Recombinant Proteins; D013455:Sulfur; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; D003545:Cysteine