"MGATP Energy Transduction in Nitrogenase"

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Seefeldt 9315835 The long range objective of this project is to define the mechanism by which MgATP hydrolysis is coupled to electron transfer and substrate reduction in the metalloenzyme complex, nitrogenase. The approach will be to use date from the recently solved x-ray crystal structures of both nitrogenase components coupled with site- directed mutagenesis and biochemical characterization of altered proteins to define the functions of specific amino acids in the mechanism of this reaction. A series of amino acids within the iron protein (FeP) components of nitrogenase have been targeted as a result of their conservation among iron proteins from 22 different organisms, their inclusion in nucleotide binding motifs and by their location in the x-ray structure of the FeP. The specific objectives for this project period will be to elucidate the function of amino acids within the FeP involved in: 1) nucleotide phosphate binding and hydrolysis, 2) adenine recognition, 3) component docking and electron transfer and 4) the MgATP induced conformational change. A system has been developed that allows the rapid generation of altered FePs by site-directed mutagenesis with expression in Azotobacter vinelandii. The altered proteins will be purified and subjected to detailed biochemical and biophysical characterizations to determine the function of the original amino acid. These characterization studies will include steady state and pre-steady state kinetic studies, analysis of conformational states, and structure determination by x-ray crystallography. Preliminary results have begun to define residues in the FeP that are essential to MgATP binding and hydrolysis and to the conformational switch that is part of the energy transduction mechanism. It is expected that the results from this project will provide a more detailed biochemical understanding of the MgATP coupled electron transfer mechanism in nitrogenase and will have broader applications to our knowledge of other nucleotide coupled energy transduction systems. Finally, the results from this study should provide a foundation of knowledge required to initiate rational protein engineering of nitrogenase to develop new properties such as altered nucleotide affinities. %%% The long range objective of this project is to define the mechanism by which MgATP hydrolysis is coupled to electron transfer and substrate reduction in the metalloenzyme complex, nitrogenase. Reduced forms of nitrogen are essential to all living organisms. The need for reduced nitrogen is especially limiting in modern agriculture where it must be supplied in the form of fertilizers. The nitrogen in fertilizers is prepared by a chemical reaction that requires large amounts of energy in the form of high temperatures and pressures. In contrast to this chemical process, many soil bacteria can convert atmospheric N2, a gas that constitutes 80% of the air that we breath, into ammonia, an agriculturally useful nitrogen source, without the requirements for high temperature or pressure. This bacterial process, called nitrogen fixation, is catalyzed by the enzyme nitrogenase. Nitrogenase catalyzed ammonia production accounts for the largest total input of fixed nitrogen into environment and represents a very attractive alternative to industrially prepared fertilizers for agriculture. We are investigating the mechanism of this biological process at the molecular level, especially the requirement for the cellular energy source MgATP. Our approach will be to combine modern genetic cloning techniques with biochemical and biophysical techniques to define the mechanism of energy utilization by nitrogenase. We anticipate that the results of our investigations will provide a detailed understanding of the mechanism of nitrogenase and will provide the foundation knowledge that will be required to initiate protein engineering of nitrogenase. Such studies could prove valuable to the future utilization of nitrogen ase in agriculture. ***