This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The biological reduction of dinitrogen to ammonia is achieved in MoFe nitrogenase at a remarkable Fe7S9MoX cluster termed FeMocofactor. Despite the availability of high resolution crystal structures, the site of substrate binding and mechanism of subsequent reduction remain unclear. Both Fe and Mo sites have been proposed as the locus of substrate chemistry, and it been suggested that the cofactor may adopt a more open conformation as part of turnover. EXAFS is clearly an ideal technique to help resolve these issues ? substrate binding should be directly observable and cofactor conformational change should be apparent through changes in ?longrange? Mo-Fe and Fe-Fe interactions. However, previous EXAFS structural studies have been limited by the inherent inability of the enzyme to be isolated in pure reduced, substrate bound states. With our collaborator Lance Seefeldt, we propose to use EXAFS to substrates bound to appropriately SGM modified MoFe nitrogenase. Work in the Seefeldt laboratory has prepared SGM variants, such as 70Ala, which, allow preparation of bound intermediate states in high yield. These currently include bound propargyl alcohol and hydrazine as well as other intermediates. In addition, with our collaborator Paul Ludden, we plan to exploit the ability of the FeMoco biosynthesis protein NafY to coordinate FeMoco, provide a small protein model of the MoFe active center. These studies should enable us to determine the whether Fe or Mo sites (or both!) coordinate substrates and intermediates and whether the FeMo-cofactor is structurally modified as part of turnover. This information should assist with the eventual elucidation of the mechanism of the environmentally important enzyme. In addition, the novel bioinorganic chemistry uncovered could well lead to the development of novel catalysts.
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