title: Biochemical characterization of iron-sulfur cluster assembly in the scaffold IscU of Escherichia coli.
abstract: Iron-sulfur cluster is one of the most common prosthetic groups, and it functions in numerous biological processes. However, little is currently known about the mechanisms of iron-sulfur cluster biosynthesis. In this study, we cloned and purified iron-sulfur cluster assembly proteins from Escherichia coli and assembled the cluster in vitro. The results showed that the assembly of iron-sulfur cluster is completed in about 20 min. Although iron or sulfur binds with IscU equivalently, 2-fold amount of iron or cysteine compared with that of IscU is better for the cluster formation, while high concentrations of IscS (IscS/IscU > 1 : 10) do not facilitate the cluster formation. Environmental pH plays an important role in iron-sulfur cluster assembly; the cluster was well assembled at pH 7.6-8.0, but was inhibited at pH less than 7.4. On supply of a catalytic amount of IscS (1/50 of IscU) and excess of other substrates, with increasing each of IscU, iron, or cysteine concentration, the iron-sulfur cluster assembly process developed from first order reaction, mixed order reaction to zero order reaction, and up to 64% of apo-IscU was converted to the [2Fe-2S] cluster-bound IscU under the optimal laboratory conditions.
authors.affiliation: College of Life Science, Zhejiang University, Hangzhou, China. wugenfu@zju.edu.cn
authors.forename: Genfu
authors.initials: G
authors.lastname: Wu
authors.forename: Lingfei
authors.initials: L
authors.lastname: Li
mesh_terms: D055162:Biocatalysis; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C487901:IscA protein, E coli; C411665:IscU protein, E coli; D011994:Recombinant Proteins; D013455:Sulfur; D007501:Iron; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase