9723715 Dooley The biochemistry, biosynthesis, structures, reactivity, and function of nitrous oxide reductase, the terminal enzyme in bacterial denitrification, is being investigated. Denitrification is a key component of the global nitrogen cycle and is the pathway that balances the cycle, returning fixed nitrogen to the atmosphere. The ecology involving nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release N20 to the atmosphere, where it may contribute to ozone depletion and global warming. This research addresses several outstanding issues in the biochemistry of denitrification, in the bacterium Achromobacter cycloclastes, with the goal of a thorough molecular understanding of this pathway. The roles of the proteins encoded by the genes nosD, F,Y,L,X, which are (or may be) required for the biosynthesis of nitrous oxide reductase, are being examined. The general strategy is to clone and over-express the soluble, periplasmic nos-coded proteins, which may be involved in the biosynthesis of the catalytic site of nitrous oxide reductase, thereby insuring sufficient material for detailed biophysical and functional studies. Site-directed mutagenesis, together with variable -temperature absorption, CD, MCD, EPR and resonance Raman spectroscopy will be used to probe the nature of the catalytic site in nitrous oxide reductase (thought to be the Cuz site), and the mechanism of reduction of nitrous oxide, including the interactions with physiological electron donors. Concerted efforts are underway to develop rapid, generally applicable schemes to purify nitrous oxide reductase, with the objective of obtaining crystals of nitrous oxide reductase suitable for x-ray diffraction analysis. Results of these experiments should be applicable to major themes in modern biochemistry such as metalloenzyme structure and function; metal ion metabolism and the biosynthesis of metalloproteins; and the recognition and acti vation of kinetically-inert small molecules. This research project seeks to understand how bacteria in soil and water convert nitrate, an important source of nitrogen for plants, into nitrogen gas, which is released to the atmosphere. A substantial fraction of the fertilizers applied to crops are wasted as a result of this conversion. The organisms responsible contain an enzyme known as nitrous oxide reductase, which plays a critical role in the overall process. A major goal of the project is to understand how this enzyme is made, its structure, and exactly how it functions. Understanding how bacteria convert nitrate to nitrogen gas could eventually lead to effective controls of this process, thereby resulting in substantial savings to farmers. It might also be possible to turn the tables and exploit this process to safely remove nitrate from drinking-water supplies, where it can be a problem.
