Enzymology of the Hydroxylamine Oxidizing System of Nitrosomonas

在土壤中，肥料氨被氧化成亚硝酸盐（NH3 + 1.502- HNO 2 +H2O）的硝化作用 欧洲亚硝化单胞菌亚硝酸盐被硝化菌氧化成 硝酸盐是非豆科植物食物蛋白质的主要氮源 作物 因此，大多数食物氮的初始转化是 由亚硝化单胞菌进行。 具有重大经济意义的是 土壤中氮的损失是由亚硝化单胞菌产生的氮气体， NH3的氧化或硝酸盐的还原 细菌 通过反硝化控制农业损失， 通过使用氨氧化抑制剂， 亚硝化单胞菌 这对提高对以下方面的认识具有重要意义： 氨氧化成亚硝酸盐的机理。 NH3被O2氧化成NH 2 OH，然后被氧化（NH 2 OH +H2O）-HNO 2 + 4 e- +4H+）通过酶羟胺 氧化还原酶“HAO”。 HAO是一个庞大而异常复杂的 血红素蛋白（7个c-血红素和1个底物结合“P460”组， 60，000 MW蛋白质）。 四个电子从HAO传递到四血红素 细胞色素c-554。两个电子然后传递到一个膜电子 转移链，最后转化为末端氧化酶， 水 另外两个电子从细胞色素c-554通过， 单独的电子转移链到氨氧化 反应 该项目的目标包括对HAO酶的分析， 细胞色素c-554和电子传递链到分子氧。 他们打算确定这两种蛋白质的结构。 将特别注意HAO的活性部位。 的 活性中心的翻转机制和电子转移 从活性部位到HAO血红素，再到细胞色素c-554的血红素 将被追捕。 该方法包括蛋白质纯化， 光谱学，肽分析，DNA测序，动力学， 硅化物衬底，电子顺磁共振光谱， 穆斯堡尔谱学和X射线晶体学分析。

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.