MODELS OF NITRATE REDUCTASES AND RELATED ENZYMES

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DESCRIPTION: (adapted from applicant's abstract) Molybdenum is an essential trace element required for all forms of life. In humans pterin-containing molybdoenzymes are directly involved in purine metabolism (xanthine oxidase, XO), and in sulfur metabolism (sulfite oxidase, SO). Defects in the 'molybdenum cofactor' synthesis, the catalytic site of these two enzymes, are fatal in children. In the nitrogen cycle, a pterin-containing molybdoenzyme, nitrate reductase (NR), catalyzes the reduction of nitrate to nitrite. Excess nitrate in water affects the health of a significant population by causing severe physiological disorders including premature termination of pregnancy. Recently, excess nitrate has been linked with non-Hodgkin's lymphoma. This proposal seeks to model certain aspects of NR. NRs are thought to catalyze direct oxygen atom transfer (OAT) reaction between a substrate and a water molecule. Theoretical studies of the OAT reaction has led to a proposal for the reaction pathway, that passes through a stable intermediate, which needs to be validated by experiment. Based on protein structures, OAT reactions involving monooxo-Mo(VI) and desoxo-Mo(IV) centers have been proposed for enzymes such as dissimilatory NR and dimethylsulfoxide reductase. To fully understand this novel reactivity, it is critical to understand the electronic structures of such centers. The structural studies also unequivocally showed that Mo-centers, which are the sites of catalysis, are buried inside the protein scaffolding. Dendritic molecules have been used to model metalloproteins such as heme and iron-sulfur proteins. The overall goal of this research is to understand the reactivity of NR by an integrated program of inorganic, physical and theoretical studies on synthetic molecules. The investigators will use discrete well-defined small molecules for answering detailed questions such as the geometry of the proposed intermediate, and synthesize large dendritic molecules to approach modeling the protein architectural features such as encapsulation. These compounds will be analyzed with a combination of techniques such as electrochemistry, optical spectroscopy, and magnetic resonance spectroscopy. The specific aims of the proposal are: 1. To understand the mechanism of the oxygen atom transfer reaction with discrete molybdenum complexes and to explore the details of the electronic structure of molybdenum (VI,IV) centers. 2. To develop dendritic systems for evaluating the effect of protein scaffolding on the properties of molybdenum centers. The investigators believe that answering these critical questions with results of the proposed research will provide a better understanding of NR and other pterin-containing molybdoenzymes as well as metalloenzymes in general.