Identification Of Cys Ligands For The [2Fe-2S] And [4Fe-4S] Clusters In Dre2 Using Electron Paramagnetic Resonance Spectroscopy:Both Clusters Are Essential For Cell Viability

Yeast Dre2?homologous to human anamorsin?is an essential component for cytosolic Fe/S cluster biosynthesis.Dre2 forms a complex with the diflavin protein Tah18.The N-terminal domain of Dre2 is of uncertain function;although it shares homology to methyltransferases,it has no activity and does not bind S-adenosyl methionine.The C-terminal domain,however,contains eight evolutionarily conserved cysteine residues,and we previously demonstrated by Electron Paramagnetic Resonance?EPR?that the yeast Dre2 overexpressed in E.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 EPR.We found that the C252A,C263A,C266A,and C268A substitutions lacked the EPR signals assigned for the[2Fe-2S]cluster,while the C314A,C322A and C325A mutants lacked the[4Fe-4S]cluster signals.The C311A mutant lacked both signals.Our data clearly suggest that the[2Fe-2S]cluster is ligated to Cys252,Cys263,Cys266,and Cys268,while 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(g_x,y,z=1.830,1.947,and 2.0181)and for the[2Fe-2S]cluster(g_x,y,z=1.919,1.962,and 2.001).We also observed spin-spin interaction between the two clusters,suggesting that these two clusters are very close to each other.Furthermore,using a yeast shuffle strain,we demonstrated for the first time that both clusters are essential for cell viability.