STRUCTURE AND MECHANISM OF FE-S PROTEINS

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The goal of our laboratory is to determine the role of protein structure in redox reactions, especially those involving Fe:S centers for electron transfer. We have chosen nitrogen reduction as a model. Although elegant spectroscopic studies have identified new Fe:S and Mo:Fe:S centers in nitrogenase proteins, we have only a limited picture of the protein structure. Yet, it is the latter which undoubtedly influences the properties of the metallo centers. For nitrogenase, no specific amino acids have been identified as part of the electron transfer processes. Only the Fe:S center ligands for the Fe-protein have been determined. It is our intention to use solution chemical methods to study changes in protein structure during catalysis and electron transfer. We will correlate the structural changes and chemical reactivity with the spectral properties of Fe:S proteins. For the proposed studies, the Fe-protein (Av2) and MoFe-protein (Av1) from Azotobacter vinelandii nitrogenase will be used. The specific objectives for the next five years are: 1. To study the interconversion of the Fe:S center in Av2 using spectral and chemical modification methods. 2. To reconstitute enzymatically active Av2 from inactive forms. 3. To identify amino acid residues in the ATP/ADP binding site and their relationship to the Fe:S center. 4. To identify potential thiol ligands of Fe:S and Mo:Fe:S centers in Avl. 5. To identify the binding regions on Av1 and Av2 for complex formation. 6. To identify potential catalytically important residues in the substrate reduction site. 7. To study the thiol reactivity in "simple", spectrally well-defined Fe:S proteins.