Reduced nitrogen is an essential component of nucleic acids and proteins and thus, all organisms require this nutrient for growth. In nature the enzyme nitrogenase is responsible for converting atmospheric dinitrogen to ammonia, a form that can be used by biological systems for biosynthesis. This structurally characterized enzyme, which is composed of two separate proteins called the Fe protein and the MoFe protein, is one of the most intriguing and complex metallobiomolecules so far isolated. The research proposed herein seeks to determine the molecular level details of the mechanism of this enzyme with emphasis on how electrons are transferred from the physiological electron donor to the substrate reduction site and the role of MgATP hydrolysis in this process. It builds on our recent demonstration that the Fe protein could be reduced to the unprecedented all ferrous [4Fe-4S]0 oxidation state and seeks to determine if the electron transfer reactions are one- electron or two-electron processes. The major approach is to construct altered forms of Fe and MoFe protein (both by site-directed mutagenesis and in vitro assembly) that are blocked for electron transfer to all substrates and that accumulate reduced forms of the metal clusters contained within these two proteins. These forms will them be characterized by X-ray crystallography, by biochemical and kinetic studies and by a variety of spectroscopic techniques with emphasis on the use of isotopic hybrids and Mossbauer spectroscopy. The information obtained will be invaluable in attempts to enhance biological nitrogen fixation or to duplicate it synthetically. In addition, the knowledge gained here is expected to greatly increase our understanding of a variety of fundamentally important processes like nucleotide controlled energy transduction, long range electron and proton transfer and multielectron transfer reactions. Finally, although metalloproteins play a variety of essential roles in catabolism, metabolic regulation and metal storage very little is known about the biosynthesis of metal centers and their incorporation into proteins. The studies proposed herein will also focus on the molecular mechanism of the insertion of one metal cluster, the iron-molybdenum cofactor, into the MoFe protein. These studies are expected to have an important impact on our understanding of the general issue of how preformed metal clusters can be inserted into proteins.
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