IscU as a scaffold for iron-sulfur cluster biosynthesis: Sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells i s known to be mediated by two highly conserved proteins, termed IscS and Is cU in prokaryotes. The homodimeric IscS protein has been shown to be a cyst eine desulfurase that catalyzes the reductive conversion of cysteine to ala nine and sulfide. In this work, the time course of IscS-mediated Fe-S clust er assembly in IscU was monitored via anaerobic anion exchange chromatograp hy, The nature and properties of the clusters assembled in discrete fractio ns were assessed via analytical studies together with absorption, resonance Raman, and Mossbauer investigations. The results show sequential cluster a ssembly with the initial IscU product containing one [2Fe-2S](2+) cluster p er dimer converting first to a form containing two [2Fe-2S](2+) clusters pe r dimer and finally to a form that contains one [4Fe-4S](2+) cluster per di mer, Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductivel y labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2) clusters in IscU are shown to have incomplete cysteinyl ligation. In addi tion, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is b est interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU sup ports the proposal that this ubiquitous protein provides a scaffold for Isc S-mediated assembly of clusters that are subsequently used for maturation o f apo Fe-S proteins.