The nitrogenase enzyme system catalyzes the ATP-dependent reduction of dinitrogen to the metabolically usable form of ammonia during biological nitrogen fixation. Nitrogenase is a prototypic example of an enzyme with multiple and varied iron-sulfur clusters that participate in electron transfer and substrate reduction, as well as providing an excellent model for energy transduction of ATP hydrolysis. The Fe-protein of nitrogenase serves as the unique, ATP hydrolyzing electron donor for substrate reduction on the MoFe-protein. We have proposed a model for the nitrogenase reaction in which the Fe-protein functions as an electron donor and as a coordinator of the electron flux within the MoFe-protein, based upon the strong structural and functional similarities between the Fe-protein and other nucleotide dependent switch proteins the redox properties of the MoFe-protein, and the conformational changes observed in the crystal structure of the complex between the Fe-protein and MoFe-protein stabilized by ADP.AlF/4-. A second role for Fe-protein is a participant in the biosynthesis of the FeMo-co-factor, a metallocluster in the MoFe-protein that is the site of substrate reduction. Although the Fe protein does not play a redox role in this process, it is an essential element and most likely serves to regulate the conformational state of the MoFe-protein during the insertion reaction. In this capacity, the Fe-protein functions much like other nucleotide switch proteins and may serve as a model for switch proteins in a variety of signal and energy transduction processes. We will utilize crystallographic, biochemical and spectroscopic approaches to investigate the enzymatic and assembly mechanisms of nitrogenase, emphasizing the multiple functions of Fe-protein and the common structural threads among nucleotide dependent switch proteins of broadly different functions. Toward these objectives, we will address: 1. the relationship between nucleotide state and protein structure, especially in Fe-protein. 2. how Fe-protein interacts with MoFe-protein during insertion of the FeMo-co-factor. 3. how electron transfer and substrate/inhibitor binding in MoFe-protein are influenced by Fe-protein. 4. the correspondence between crystallographically defined metallocluster structures and spectroscopically assigned oxidation states in the Fe-protein and MoFe-protein.
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