All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys؉ cluster of C42D FdI exhibits only an S ؍ 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.A fundamental question in [Fe-S] protein biochemistry concerns how cysteine ligand and neighboring residue organization determines [Fe-S] cluster type, and whether or not one type can be converted to another (for reviews, see Refs. 1-9). These issues are important, in part, because there are physiological situations where 3Fe to 4Fe or 4Fe to 2Fe cluster interconversion reactions modulate the activity of an enzyme or a regulatory protein (1, 8 -13 2ϩ/ϩ cluster, is proving to be an excellent model system for elucidating the properties of these redox centers. As shown in Fig. 2, the amino acid sequence Cys-X-X-Cys-X-X-Cys, which provides three of the four cysteine ligands to a [4Fe-4S] 2ϩ/ϩ cluster, is a very common motif. Interestingly, with one exception, all naturally occurring ferredoxins or ferredoxin variants that contain 4Fe clusters, which can be easily interconverted with 3Fe clusters have instead a Cys-X-X-Asp-X-X-Cys motif. The Asp serves as the ligand that is lost during the cluster interconversion process (23,(25)(26)(27)(28)(29). The exception is DgFdII where the central Cys is covalently modified in the [3Fe-4S] cluster-containing state and where the interconversion reaction is complicated by a change in subunit composition (10, 30). Aconitase, which also easily interconverts, also has a non-Cys (water or OH Ϫ ) ligand (31,32). In contrast, for ferredoxins or variants that contain 2ϩ/ϩ clusters with four Cys ligands, attempts to convert the 4Fe center to a 3Fe center more often produce only poor yields or result in complete degradation accompanied by denaturation (33)(34)(35)(36)(37)(38). This is certainly the case for the protein of interest in this study, AvFdI, where the oxidative destruction of its 2ϩ/ϩ cluster has been studied in some detail (38 -40). In this study, we changed the central cysteine of the Cys 39 -X-X-Cys 42 -X-X-Cys 45 motif, which coordinates the [4Fe-4S] 2ϩ/ϩ cluster, into an aspartate. This paper describes the redox and spectroscopic properties of the new 2ϩ/ϩ cluster, and our various attempts to convert it into [3Fe-4S] ϩ/0 . It also is the first report of the x-ray structure of an aspartate-liganded 2ϩ/ϩ cluster.