DELTA-T14 DELTA-D15 AZOTOBACTER-VINELANDII FERREDOXIN-I - CREATION OFA NEW CYSXXCYSXXCYS MOTIF THAT LIGATES A [4FE-4S] CLUSTER/

In clostridial-type ferredoxins, each of the two [4Fe-4S](2+/+) cluste rs receives three of its four ligands from a CysXXCysXXCys motif, Azot obacter vinelandii ferredoxin I (AvFdI) is a seven-iron ferredoxin tha t contains one [4Fe-4S](2+/+) cluster and one [3Fe-4S](+/0) cluster. D uring the evolution of the 7Fe azotobacter-type ferredoxins from the 8 Fe clostridial-type ferredoxins, one of the two motifs present changed to a CysXXCysXXXXCys motif, resulting in the inability to form a 4Fe cluster and the appearance of a 3Fe cluster in that position. In a pre vious study, we were unsuccessful in using structure as a guide in des igning a 4Fe cluster in the 3Fe cluster position of AvFdI. In this stu dy, we have reversed part of the evolutionary process by deleting two residues between the second and third cysteines. UV/ Vis, CD, and EPR spectroscopies and direct electrochemical studies of the purified prot ein reveal that this Delta T14/Delta D15 FdI variant is an 8Fe protein containing two [4Fe-4S](2+/+) clusters with reduction potentials of - 466 and -612 mV versus SHE. Whole-cell EPR shows that the protein is p resent as an 8Fe protein in vivo. These data strongly suggest that it is the sequence motif rather than the exact sequence or the structure that is critical for the assembly of a 4Fe cluster in that region of t he protein. The new oxygen-sensitive 4Fe cluster was converted in part ial yield to a 3Fe cluster. In known ferredoxins and enzymes that cont ain reversibly interconvertible [4Fe-4S](2+/+) and [3Fe-4S](+/0) clust ers, the 3Fe form always has a reduction potential ca. 200 mV more pos itive than the 4Fe cluster in the same position. In contrast, for Delt a T14/Delta D15 FdI, the 3Fe and 4Fe clusters in the same location hav e extremely similar reduction potentials.