Iron sulfur (Fe-S) proteins contain clusters composed predominately of Fe, S2- and cysteine residues. These proteins are ubiquitous occuring in all known organisms and are central to every essential biological process. Three types of clusters containing respectively, 1, 2, and 4 Fe atoms are now well characterized and recently clusters containg three Fe atoms have been discovered. The first three exhibit multiple oxidation levels. It has recently been shown that electron transport is not the sole function of these proteins. It is now abundantly clear that the variety of Fe-S cluster structures is greater than previously recognized and continues to expand. The chemistry is far more extensive than simple, reversible one-electron redox behavior. The goal of this proposal is to ascertain how the primary sequence determines which type of cluster is functional in a given protein. To achieve this goal the structures and reactivities of protein-bound 3 FE and 4 FE (Fe-S) clusters in A. vinelandii fereedoxin I (AVFD) will be studied in depth. This small protein is ideally suited for such an investigation, as it expresses multiple forms and X- ray data is becoming available. Consequently, it is planned to purify and crystallize the three new forms of AvFdI, characterize them physico-chemically, modify them by site directed mutagenesis, and purify and characterize the resulting fereedoxins. This work may prove crucial to our quest to understand nitrogenase and other multicentered redox proteins containing Fe-S centers. Support is strongly recommended with a high priority.