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[摘要]：Hydroxylamine oxidoreductasc (HAO) from Nitrosomonas europaea normally catalyzes oxidation of NH2OH to NO2-. This paper reports experiments in which HAO was thermodynamically poised to catalyze reduction of NO2- to NH4+. HAO was found to catalyze the reduction of NO2- by methyl viologen monocation radical (MVred), displaying a hyperbolic dependence on NO2- concentration, with a k(cat1) of 6.8 +/- 0.3 s(-1) and a K-m1 of 7.6 +/- 0.9 mM. HAO also catalyzed the reduction of NH2OH by MVred, with a hyperbolic dependence on NH2OH concentration, and a k(cat2) of 245 +/- 3 s(-1) and a K-m2 of 6.8 +/- 0.2 mM (k(cat1) and k(cat2) reflect the maximum number of electrons transferred from MVred per second). We had previously demonstrated that HAO catalyzes the reduction of NO by MVred to yield first NH2OH and then NH4+. Thus, overall, HAO is seen to act like a cytochrome c nitrite reductase, which catalyzes the six-electron reduction of NO2- to NH4+ by MVred. In the presence of Ru(NH3)(2+) (Ru-II) and Ru(NH3)(3+) (Ru-III) at ratios exceeding 200:1, HAO exhibited no detectable Ru-II-NO2- oxidoreductase activity, though such activity is thermodynamically favored. On the other hand, HAO could still catalyze the oxidation of NH2OH to NO by Ru-III under these conditions. The oxidative process exhibited a hyperbolic dependence on NH2OH concentration, with a k(cat3) of 98 +/- 3 s(-1) and a K-m3 of 5.2 +/- 0.8 mu M. Finally, HAO was found not to catalyze the disproportionation of NH2OH, despite the thermodynamic favorability of such a process, and the apparent opportunity presented by the HAO structure. Mechanisms are proposed to explain all the kinetic data. |
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