The broad, long term goal of the project is to gain structural and mechanistic insights into the role of MgATP in nitrogenase catalysis. Nitrogenase is a complex metal-containing enzyme that catalyzes the conversion of nitrogen gas to ammonia. During nitrogenase catalysis, the iron protein and molybdenum-iron protein associate and dissociate in a manner resulting in the hydrolysis of two molecules of MgATP and the transfer of at least one electron to the MoFe protein. Multiple cycles of iron protein association and dissociation, MgATP hydrolysis, and electron transfer are required for the complete reduction of a single molecule of nitrogen to ammonia. There are a number of aspects of nitrogenase structure/function that are interesting areas of fundamental research. Nitrogenase can be considered an ideal model system for the study of the complex metal cluster mediated catalysis, electron transfer, complex metal cluster assembly, protein-protein interactions, and nucleotide dependent signal transduction. In addition, the involvement of MgATP in nitrogenase catalysis is similar to the role of nucleotides in a large class of nucleotide binding proteins that couple nucleotide binding and hydrolysis to protein conformational changes transduced within a macromolecular assembly. Members of the class include G proteins, Ras p21, RecA, elongation factor Tu, myosin, and transducin, making the role of MgATP binding and hydrolysis one of the most fascinating aspects of nitrogenase research. We have recently been able to determine the structure of a single deletion mutant of the nitrogenase Fe protein that provides a structural mimic of the MgATP bound state. The structural insights described in the preliminary results section of the proposal provide the firmest foundation described to date for generating hypotheses concerning MgATP dependent conformational change in the Fe protein and the initial component protein interactions that trigger MgATP hydrolysis. The proposed studies apply a combined approach involving structure determination by x-ray diffraction methods and site-specific amino acid substitution experiments to gain insights into nucleotide dependent conformational change, macromolecular complex formation, and the specific protein-protein interactions occurring upon complex formation that initiate MgATP hydrolysis in nitrogenase.


John Peters