STRUCTURAL CHARACTERIZATION OF NIFB-CO

University Of Wisconsin Madison, Madison WI

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Abstract

The long term objective of this research proposal is the attainment of an understanding of iron and sulfur metabolism in general through the study of the metabolism these elements in diazotrophic bacteria. A key question in biochemistry is "How do cells synthesize the building blocks of their macromolecules?" The iron-molybdenum cofactor (FeMo-co) of dinitrogenase is the most complex biological iron-sulfur cluster known, and it's structure has yet to be duplicated using synthetic inorganic methods. An understanding of the mechanism of construction of this complex cofactor relies on an understanding of the structure and properties of its iron and sulfur precursor, NifB-co. A number of illnesses are caused by disfunctional iron metabolic pathways, and any understanding of iron metabolism gained from the study of this microbial system should assist in the understanding of similar systems in higher organisms, including humans. Recently molybdenum-cofactor deficiency was described in human subjects with hypouricemia. This study should provide insights into the form of molybdenum necessary for incorporation cofactors, and thus illucidate certain aspects of molybdenum metabolism. Therefore, the structure and properties of NifB-co will be examined. The stability of NifB-co in various organic solvents will be established. Electrochemistry will be used to determine the redox potential(s) of NifB-co, and provide paramagnetic samples EPR analysis. The ability of NifB-co in various redox states to bind heterometals or exogenous ligands will be assessed. 57Fe labelled samples of NifB-co will be prepared for Mossbauer analysis. Samples of NifB-co with iron and/or sulfur atoms isotopically labelled will be analyzed by mass spectroscopy to determine the number of irons and sulfurs present. All practicable samples of modified NifB-co generated by and for the above techniques will be tested for viability as a iron and sulfur source for FeMo-co by an established assay procedure.