Enzymology of the Hydroxylamine Oxidizing System of Nitrosomonas

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In soils, fertilizer ammonia is oxidized to nitrite (NH3 +1.502 - HNO2 +H2O) 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. Thus the initial 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 lends significance to improved knowledge of the mechanism of oxidation of ammonia to nitrite. NH3 is oxygenated by O2 to NH2OH which is then oxidized (NH2OH +H2O)- HNO2 + 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 60,000 MW protein). Four electron pass from HAO to a tetraheme cytochrome c-554. Two electrons then pass to a membrane electron transfer chain and finally to a terminal oxidase, producing water. Two additional electrons pass from cytochrome c-554 in a separate electron transfer chain to the ammonia oxidizing reaction. Goals of the project include an analysis of the enzyme HAO, the cytochrome c-554 and the electron transport chain to dioxygen. They intend to determine the structure of the two proteins. Particular attention will be paid to the active site of HAO. The mechanism of turnover in the active site and electron transfer from active site to HAO hemes and then hemes of cytochrome c-554 will be pursued. The approach involves protein purification, optical spectroscopy, peptide analysis, DNA sequencing, kinetics, suicide substrates, electron paramagnetic resonance spectroscopy, Mossbauer spectroscopy and X-ray crystallographic analysis.