Enzymology of the Hydroxylamine Oxidizing System of Nitrosomonas

在土壤中，化肥氨氧化成亚硝酸盐(NH3+1.502--HNO2+H20)被化学自养硝化细菌NitroSomonas uropaea氧化。亚硝酸盐被硝酸杆菌氧化成硝酸盐，这是非豆类作物中N的主要来源。因此，大多数食物氮素的初始转化都是由亚硝化单胞菌进行的。在经济上具有重要意义的是土壤中氮的损失，因为亚硝化单胞菌在NH3氧化过程中产生的氮气体或反硝化细菌还原硝酸盐产生的氮气体。通过使用亚硝化单胞菌的氨氧化抑制剂来尝试通过反硝化来控制农业损失。这对提高对氨氧化机理的认识具有重要意义。NH3被O2氧化成NH_2OH，然后被羟色胺氧化还原酶“HAO”氧化(NH_2OH+H_2)--HNO_2+4E-+4H+。Hao是一种大的(200kD)和异常复杂的血红蛋白(24个c血红素和3个底物结合的“p460”基团)。两个电子在电子传递链中从HA0传递到膜终端氧化酶，该链包括四氢血红素细胞色素C554和其他未知成分。另外两个电子从HAO传递到氨单加氧酶。该项目的目标包括：确定HAO的亚基结构和氨基酸序列，确定HAO的p460环结构和底物结合部位的结构，鉴定HAO的电子退出部位；确定细胞色素c-554的氨基酸序列和电子进出部位；和HAO与末端氧化酶的重组。该方法涉及到酶学、蛋白质化学、光学和EPR光谱快速动力学、克隆和DNA测序。

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.