Active Site Structure and Function of Copper Enzymes in Denitrification

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The principal goal of the proposed research is a fundamental understanding of the structural and catalytic roles played by the metal ions and protein moieties in nitrite and nitrous oxide reductases. Extensive spectroscopic and chemical studies on these reductases are proposed. These classes of multicopper redox enzymes provide new opportunities to investigate structure/function relationships in copper-containing enzymes that are involved in denitrification. Dr. Dooley will also address questions concerning the possible regulation of theses enzymes by intermediates in the denitrification pathway. In addition a partially copper-depleted form of N2O reductase, isolated from a mutant strain of Pseudomonas stutzeri, will be thoroughly characterized. The spectroscopic properties of Cu(II) will be exploited as a built-in probe of the active site by variable temperature X-ray absorption spectroscopy circular dichroism, magnetic circular dichroism resonance Raman, and EPR spectroscopy. Dr. Dooley plans to develop a N2O-specific electrode assay for activity which would facilitate kinetics and mechanism studies of N2O reductase. Effects of exogenous ligands (inhibitors, denitrification intermediates, substrate analogs) and pH on the spectral properties and kinetics of the copper reductases will also be investigated. Integrating results from spectroscopic experiments and mechanistic studies will permit new structure/reactivity/function correlations among multicopper oxidases and reductases to be formulated. Understanding the relationships between the structures and mechanisms of nitrite and N2O reductases is expected to play an essential role in developing rational strategies to control and exploit biological denitrification.