SITE-DIRECTED MUTAGENESIS AZOTOBACTER-VINELANDII FERREDOXIN-I - CYSTEINE LIGATION OF THE [4FE-4S] CLUSTER WITH PROTEIN REARRANGEMENT IS PREFERRED OVER SERINE LIGATION

The [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I receives t hree of its four ligands from a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence a t positions 39-45 while the fourth ligand, Cys(20), is provided by a d istal portion of the sequence. Previously we reported that the site-di rected mutation of Cys(20) to Ala (C20A protein) resulted in the forma tion of a new [4Fe-4S] cluster that obtained its fourth ligand from Cy s(24), a free cysteine in the native structure. That ligand exchange r equired significant protein rearrangement, Here we report the conversi on of Cys(20) to Ser (C20S protein), which gives the protein the oppor tunity either to retain the native structure and use the Ser(20) O-gam ma as a ligand or to rearrange and use Cys(24). X-ray crystallography demonstrates that the cluster does not use the Ser(20) O-gamma as a li gand; rather it rearranges to use Cys(24). I, the C20S protein the [4F e-4S] cluster has altered stability and redox properties relative to e ither C20A or the native protein.