EPR studies of wild type and mutant Dre2 identify essential [2Fe–-2S] and [4Fe–-4S] clusters and their cysteine ligands

Yeast Dre2 (anamorsin or CIAPIN1) is an essential component for cytosolic Fe/S cluster biosynthesis. The C-terminal domain contains eight evolutionarily conserved cysteine residues, and we previously demonstrated that the yeast Dre2 overexpressed in Escherichia coli contains one binuclear ([2Fe–2S]) cluster and one tetranuclear ([4Fe–4S]) cluster. In this study, we replaced each conserved cysteine with alanine and analyzed the effects by Electron Paramagnetic Resonance. Although the C311A mutant lacked both signals, our data clearly suggest that the [2Fe–2S] cluster is ligated to Cys252, Cys263, Cys266 and Cys268, whereas the [4Fe–4S] cluster is ligated to Cys311, Cys314, Cys322 and Cys325. By simulation analysis of the C263A and C322A data, we obtained the g-values for the [4Fe–4S] cluster (gx,y,z = 1.830, 1.947 and 2.018) and for the [2Fe–2S] cluster (gx,y,z =1.919, 1.962 and 2.001). We also observed spin–spin interaction between the two clusters, suggesting their close proximity. Chemically reconstituted Dre2 showed air sensitivity of the [4Fe–4S] cluster converting to a [2Fe–2S] cluster. Furthermore, using a yeast shuffle strain, we demonstrated for the first time that each of the Cys Fe–S cluster ligands with the exception of C252 is essential, indicating that both Dre2 clusters are needed for cell viability.