In clostridial-type ferredoxins, each of the two [4Fe-4S]2+/+ clusters receives three of its four ligands from a CysXXCysXXCys motif. Azotobacter vinelandii ferredoxin I (AvFdI) is a seven-iron ferredoxin that contains one [4Fe-4S]2+/+ cluster and one [3Fe-4S]+/0 cluster. During the evolution of the 7Fe azotobacter-type ferredoxins from the 8Fe 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 previous study, we were unsuccessful in using structure as a guide in designing a 4Fe cluster in the 3Fe cluster position of AvFdI. In this study, 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 protein reveal that this DeltaT14/DeltaD15 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 present 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 the protein. The new oxygen-sensitive 4Fe cluster was converted in partial yield to a 3Fe cluster. In known ferredoxins and enzymes that contain reversibly interconvertible [4Fe-4S]2+/+ and [3Fe-4S]+/0 clusters, the 3Fe form always has a reduction potential ca. 200 mV more positive than the 4Fe cluster in the same position. In contrast, for DeltaT14/DeltaD15 FdI, the 3Fe and 4Fe clusters in the same location have extremely similar reduction potentials.