Denitrification is a key process in the nitrogen cycle, which controls the amount of nitrogen available for plant growth. It is estimated that up to 30% of fertilizer nitrogen is lost via denitrification. The pathway by which nitrate is reduced to gaseous nitrogen is still not known in detail, but nitrite and nitrous oxide have been identified as free intermediates, and there appear to be two routes from NO2 - to N2O, one direct and the other via NO. The object of the proposed research is to elucidate the pathway(s) by which NO2 - is converted to N2O. The major questions to be answered are how NO2 - is reduced by the heme cd1 and copper-containing nitrite reductases, how NO is reduced to N2O by nitric oxide reductase, and what factors control the relative flux via the two routes from NO2 - to N2O in vivo. A multifaceted approach to the problem is described, combining chemical and biological studies, and focussing on the mechanism by which the N-N bond of N2O is formed. Specific techniques to be employed include isotopic labelling experiments; kinetics studies; cryoenzymological and spectroscopic studies of the purified enzymes; and electron microscopy. Specific systems to be examined include whole cells and cell-free extracts of selected wild-type denitrifying bacteria and appropriate mutants, as well as purified nitrite reductase and crude nitric oxide reductase preparations.
