Iron-sulfur cluster biosynthesis: characterization of IscU-IscS complex formation and a structural model for sulfide delivery to the [2Fe-2S] assembly site

Recent work on the bacterial iron-sulfur cluster (isc) family of gene products, and eukaryotic homologs, has advanced the molecular understanding of cellular mechanisms of iron-sulfur cluster biosynthesis. Members of the IscS family are pyridoxyl-5'-phosophate dependent proteins that deliver inorganic sulfide during assembly of the [2Fe-2S] cluster on the IscU scaffold protein. Herein it is demonstrated through calorimetry, fluorescence, and protein stability measurements that Thermotoga maritima IscS forms a 1:1 complex with IscU in a concentration-dependent manner (K (D) varying from 6 to 34 mu M, over an IscS concentration range of approximately 2-50 mu M). Docking simulations of representative IscU and IscS proteins reveal critical contact surfaces at the N-terminal helix of IscU and a C-terminal loop comprising a chaperone binding domain. Consistent with the isothermal titration calorimetry results described here, an overall dominant contribution of charged surfaces with a change in the molar heat capacity of binding, Delta C (p) similar to 199.8 kcal K-1 mol(-1), is observed that accounts for approximately 10% of the total accessible surface area at the binding interface. Both apo and holo IscUs and homologs were found to bind to IscS in an enthalpically driven reaction with comparable K (D) values. Both helix and loop regions are highly conserved among phylogenetically diverse organisms from a pool of archael, bacterial, fungal, and mammalian representatives.