Iron-sulfur ([Fe-S]) proteins are ubiquitous, occurring in all organisms from the most primative archaebacteria to the most advanced eucaryotes. They are not only central to almost every essential biological redox process but they also catalyze hydration/dehydration reactions, participate in DNA repair and serve important roles in eucaryotic gene expression. The general goal of the work proposed here is to elucidate the interrelationships between protein structure and [Fe-S] cluster structure, function, redox properties and reactivity. To meet these goals we are using site-directed mutagenesis to modify the amino acid sequence of our model protein, Azotobacter vinelandii FdI. Following the expression of the mutated proteins in their native background they will be purified in large quantities and their properties will be completely characterized by X-ray crystallographic, biochemical, spectroscopic and electrochemical methods. The chemical behavior of these new forms of AvFdI, including their conversion to so far unknown forms of AvFdI will also be characterized. In addition, we will determine whether or not the new mutant forms of AvFdI are functional in vivo and will probe both the electron transfer and possible regulatory roles of the protein. This work constitutes the first application of site-directed mutagenesis to an [Fe- S] protein of known structure. The results will be applicable to the entire class of [Fe-S] proteins and can be expected to yield much new information relevant to the identification of [Fe-S] cluster structures in more "complex" [Fe-S] proteins yet to be characterized.