<i>In vivo</i> iron–sulfur cluster formation

Abstract

It has been proposed that iron-sulfur [Fe-S] clusters destined for the maturation of [Fe-S] proteins can be preassembled on a molecular scaffold designated IscU. In the present article, it is shown that production of the intact Azotobacter vinelandii [Fe-S] cluster biosynthetic machinery at levels exceeding the amount required for cellular maturation of [Fe-S] proteins results in the accumulation of: (i) apo-IscU, (ii) an oxygen-labile [2Fe-2S] cluster-loaded form of IscU, and (iii) IscU complexed with the S-delivery protein, IscS. It is suggested that these species represent different stages of the [Fe-S] cluster assembly process. Substitution of the IscU Asp(39) residue by Ala results in the in vivo trapping of a stoichiometric, noncovalent, nondissociating IscU-IscS complex that contains an oxygen-resistant [Fe-S] species. In aggregate, these results validate the scaffold hypothesis for [Fe-S] cluster assembly and indicate that in vivo [Fe-S] cluster formation is a dynamic process that involves the reversible interaction of IscU and IscS.