Enzymology of the Hydroxylamine Oxidizing System of Nitrosomonas

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In soils, fertililzer ammonia is oxidized to nitrite (NH3+1.5 02--HNO2+H20) by the chemautotrophic 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 ntirogen 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 ammonia oxidation. NH3 is oxygenated by O2 to NH2OH which is then oxidized (NH2OH+H2)--HNO2+4e-+4H+) by the enzyme hydroxcylamine oxidoreductase, "HAO." HAO is a large ( 200 Kd) and unusually complex hemoprotein ( 24 c hemes and 3 substrate binding "P460" groups). Two electrons pass from HAO, in an electron transport chain which includes the tetraheme cytochrome c554 and other unknown components, to a membrane terminal oxidase. Two additional electrons pass from HAO to the ammonia monoxygenase. Goals of the project include: determination of the subunit structure and amino acid sequence of HAO, the ring structure of P460 of HAO and the structure of the substrate binding site and identification of the electron exit site of HAO; determination of the amino acid sequence and electron entry and exit sites of cytochrome c- 554; and reconstitution of HAO with the terminal oxidase. The approach involves enzymology, protein chemistry, optical and EPR spectroscopy rapid kinetics, cloning and DNA sequencing.