The project focuses on the metabolism and enzymology of nitrogen oxides, chiefly but not exclusively in denitrifying bacteria. Recent success in the purification of nitric oxide and nitrous oxide reductases from Paracoccus denitrificans and of a new type of nitrous oxide reductase (cytochrome c- containing) from Wolinella succinogenes permits further detailed characterization of these enzymes, purification of the analogous enzymes from other bacteria for comparison, and raising of antibodies to test for antigenic similarities across species. Structure and function will be determined for the chromophoric lipid associated with nitric oxide reductase. The structural gene for nitric oxide reductase will be cloned and sequenced. The kinetics of NO uptake and production and the steady-state levels of NO will be studied in denitrifying and enteric bacteria with use of a recently devised NO-electrode and hemoglobin trapping methods. These studies are directed at resolving the question whether NO is an intermediate in denitrification and exploring the generality and enzymology of NO production from nitrite by enteric bacteria. The mechanism of enzyme-catalyzed nitrosations and the possible participation of NO in these reactions will be studied with use of nucleophiles that yield a nongaseous nitrosylated product, e.g., morpholine and 2,3- diaminonoaphthalene. The ability of certain bacteria to reduce nitrogen oxides to nitrogen was described in the last century and termed denitrification. The process is energy yielding and represents an alternative to aerobic respiration. Denitrification is of considerable importance geochemically, because it represents the major route for return of fixed nitrogen to the atmosphere as N2. It therefore must serve in the mechanisms which control the amount of oxygen the earth's atmosphere. It is clear that the biochemical mechanisms of denitrification are not as well understood as those of some other areas of nitrogen metabolism. A detailed understanding of these mechanisms might lead to inhibitors of denitrification in agriculture and the facilitation of N2 production in, for example, sewage treatment.***//
