STUDIES OF HYDROXYLAMINE METABOLISM OF NITROSOMONAS EUROPAEA .I. PURIFICATION OF HYDROXYLAMINE OXIDASE

The chemoautotrophic bacterium Nitrosomonas europaea derives its energy from the oxidation of ammonia and hydroxylamine. Short-term incubation experiments with intact cells reveal that both substrates are quantitatively oxidized to nitrite. When the structural integrity of the cell is destroyed, all ability to oxidize ammonia is lost, and only 10% of the hydroxylamine-oxidizing specific activity remains. Moreover, not more than 30-40% of the total hydroxylamine metabolized by crude cell-free extracts of Nitrosomonas is converted to the expected physiological product, nitrite. Efforts to obtain cell-free ammonia-oxidizing activity have failed. However, very active hydroxylamine oxidation occurs in the presence of added terminal electron acceptor compounds. Again, only a fraction of the hydroxylamine utilized can be accounted for as nitrite. Maximum nitrite formation occurs in the presence of phenazine methosulfate. The enzyme responsible for hydroxylamine oxidation, hydroxylamine oxidase, has been isolated in highly purified form by the sequence of steps: ultracentrifugation → sucrose density gradient centrifugation →- Sephadex G-200 chromatography. Although only 3040% of the total oxidized hydroxylamine is converted to nitrite by crude extracts, this number rises progressively as the enzyme is isolated and reaches 70% for the purified enzyme. The improved stoichiometry is at least in part accounted for by the removal of a soluble cytochrome c552 containing material (molecular weight approximately 20,000 g/mole) which has been denoted as fraction F2. This substance specifically inhibits the formation of nitrite by hydroxylamine oxidase. It has no effect on either the rate or extent of hydroxylamine oxidation. Hydroxylamine oxidase is the initial acceptor of electrons from hydroxylamine. The enzyme is unable to ultimately transfer electrons to molecular oxygen, however, and this function is performed by the terminal oxidase of the cell. Evidently, a marked reduction in the efficiency of sequential electron transfer accompanies cellular lysis. The striking stimulation of hydroxylamine oxidase activity by added terminal electron-acceptor compounds is in keeping with this observation. © 1968, American Chemical Society. All rights reserved.