Nucleotide-Dependent Energy Transduction in Nitrogenase

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The long-term goal of this project is to define the mechanism of energy coupling (e.g., ATP hydrolysis) to N2 reduction in the metalloenzyme complex nitrogenase. The availability of fixed forms of nitrogen (e.g., ammonia) is essential to all living organisms, where it is used to make proteins, DNA, and a range of other biomolecules. The reduction of N2 from air to ammonia represents the largest input of bioavailable nitrogen in the biosphere, with biological nitrogen fixation accounting for most of the N2 reduction. Biological nitrogen fixation occurs in a large number of microorganisms that can be called diazotrophs, being catalyzed by a highly conserved metalloenzyme called nitrogenase. The specific objective of this research program is to address one of the significant unknowns about the nitrogenase mechanism, namely understanding how MgATP binding and hydrolysis are coupled to N2 reduction. Progress over recent years has revealed that nucleotide binding and hydrolysis control several steps in the nitrogenase mechanism including electron transfer from the Fe protein component to the MoFe protein component, substrate reduction on the MoFe protein, and dissociation of the two component proteins following each electron transfer event. Importantly, it is clear that nucleotides exert their influence at a distance through long-range protein conformational changes. In this project, three outstanding questions about how nucleotides participate in the nitrogenase mechanism will be considered: 1) What are the contributions of two peptide stretches within the Fe protein (termed switches I and II) in communicating from the nucleotide binding sites to influence the MoFe protein? 2) How do nucleotides regulate electron transfer from the Fe protein to the MoFe protein? 3) What are the individual contributions of two MgATP binding and hydrolysis events to the overall nitrogenase mechanism? A multidisciplinary approach will be used that utilizes x-ray structural information, in conjunction with site-directed mutagenesis and biochemical and spectroscopic methods, to unravel details of the nucleotide mechanisms. It is expected that the results from this project will contribute to a detailed understanding of the functions of nucleotides in the nitrogenase mechanism and will have broader implications in understanding the mechanisms of other nucleotide-coupled energy transduction proteins.