Hooper 9316906 In soils, fertilizer ammonia is oxidized to nitrite (NH3 + 1.5 02-> HNO2 + H2) by the chemoautotrophic nitrifying bacterium Nitrosomonas europaea.. Nitrite is oxidized by Nitrobacter to nitrate, a primary source of N for food protein in non-leguminous crops. Of great economic transformation of most food nitrogen is carried out by Nitrosomonas. Of great economic importance is loss of N in soils as N-gases produced by Nitrosomonas during oxidation of NH3 or by reduction of nitrate by denitrifying bacteria. Control of agricultural loss by denitrification is attempted by the use of inhibitors of ammonia oxidation by Nitrosomonas. This gives significance to improved knowledge of the mechanism of oxidation of ammonia. NH3 is oxygenated by O2 to NH2OH which is then oxidized (NH2OH + H20-> HN02 + 4e- + 4H+) by the enzyme hydroxylamine oxidoreductase "HAO". HAO is a large and unusually complex hemoprotein (7 c-hemes and 1 substrate binding. "P460" group per 63 kDa protein). Four electron pass from HAO to a tetraheme cytochrome c-554. Two electrons pass from cytochrome c-554 in a separate electron transfer chain to the ammonia oxidizing reaction. Two additional electrons pass to a membrane terminal oxidase, a dissimilatory nitrite reductase (during growth at low oxygen), to an ATP- dependent NAD reductase or a peroxidase (to scavange peroxide). The goal of this project is the analysis of the enzyme HAo. We intend to determine details of the structure of HAO with particular attention to the active site and mechanism of turnover. The approach involves optical spectroscopy, peptide analysis, kinetics, suicide substrates, electron paramagnetic resonance spectroscopy, Mossbauer spectroscopy and X-ray crystallographic analysis. %%% The soil and water bacterium, Nitrosomonas, is critical to the cycle of nitrogen in nature, to the production of a greenhouse and ozone destroying gas, to nitrogen availability in agriculture and in the degradation of chlori nated pollutants. This project is involved in the study of the structure of the biological catalyst which enables the organism to perform those roles. The enzyme oxidizes one of the nitrogenous intermediates, hydroxylamine, to nitrite. Is called hydroxylamine oxidoreductase. We intend to complete the determination of the structure of the enzyme. Then, by studying changes in the color of groups on the enzyme and by measuring the production of intermediate compounds by chemical means, we will find out how it is able to act as a catalyst for this unusual reaction. ***
