title: Cys-328 of IscS and Cys-63 of IscU are the sites of disulfide bridge formation in a covalently bound IscS/IscU complex: implications for the mechanism of iron-sulfur cluster assembly.
abstract: IscS and IscU from Escherichia coli cooperate with each other in the biosynthesis of iron-sulfur clusters. IscS catalyzes the desulfurization of L-cysteine to produce L-alanine and sulfur. Cys-328 of IscS attacks the sulfur atom of L-cysteine, and the sulfane sulfur derived from L-cysteine binds to the Sgamma atom of Cys-328. In the course of the cluster assembly, IscS and IscU form a covalent complex, and a sulfur atom derived from L-cysteine is transferred from IscS to IscU. The covalent complex is thought to be essential for the cluster biogenesis, but neither the nature of the bond connecting IscS and IscU nor the residues involved in the complex formation have been determined, which have thus far precluded the mechanistic analyses of the cluster assembly. We here report that a covalent bond is formed between Cys-328 of IscS and Cys-63 of IscU. The bond is a disulfide bond, not a polysulfide bond containing sulfane sulfur between the two cysteine residues. We also found that Cys-63 of IscU is essential for the IscU-mediated activation of IscS: IscU induced a six-fold increase in the cysteine desulfurase activity of IscS, whereas the IscU mutant with a serine substitution for Cys-63 had no effect on the activity. Based on these findings, we propose a mechanism for an early stage of iron-sulfur cluster assembly: the sulfur transfer from IscS to IscU is initiated by the attack of Cys-63 of IscU on the Sgamma atom of Cys-328 of IscS that is bound to sulfane sulfur derived from L-cysteine.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Kato
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Mihara
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kurihara
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Umechiyo
authors.initials: U
authors.lastname: Tokumoto
authors.forename: Tohru
authors.initials: T
authors.lastname: Yoshimura
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Esaki
mesh_terms: D000409:Alanine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D013437:Carbon-Sulfur Lyases / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007501:Iron / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008190:Lyases / Q000378:metabolism; D008956:Models, Chemical; D011485:Protein Binding; D013455:Sulfur / Q000737:chemistry*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C411665:IscU protein, E coli; D013455:Sulfur; D007501:Iron; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; D003545:Cysteine; D000409:Alanine