Biosynthesis, Structure, Function and Regulation of Nitrous Oxide Reductase

Montana State University, Bozeman MT

Investigators

Abstract

The project addresses basic questions regarding the mechanism of assembly of the novel copper sites in the enzyme nitrous oxide reductase, and their roles in catalysis. Proteins encoded by the nos cluster genes nosD,F,Y,L, which are required for the biosynthesis of nitrous oxide reductase, will be purified and characterized. The investigator's hypothesis is that one or more of the Nos proteins are involved in the assembly of the novel copper cluster that is the catalytic site in nitrous oxide reductase. Over-expression methods for nitrous oxide reductase and other nos proteins have been developed. Site-directed mutagenesis, together with spectroscopic and kinetics methods, will be used to probe the structure, bonding, and reactivity of the electron-transfer (CuA) and catalytic sites. Structural studies (by NMR or x-ray crystallography) of the proteins are planned. The results should be applicable to several major themes in modern biochemistry such as metalloprotein structure and function, the assembly of metal ion clusters in proteins, and the recognition and activation of kinetically-inert small molecules in metabolic pathways. The enzyme that is the focus of the research is the terminal enzyme in the denitrification pathway of bacteria. The denitrification pathway is a part of the global nitrogen cycle and balances the cycle by returning fixed nitrogen to the atmosphere. Global agricultural productivity and water quality are directly affected by the biological processes of nitrogen fixation, assimilation by organisms, and denitrification. Furthermore, under some circumstances denitrification may release nitrous oxide to the atmosphere, thereby contributing to ozone depletion and global warming. Consequently, it is very important to understand exactly how organisms carry out denitrification, and how to control this process.