| pubmed-article:20812758 | pubmed:abstractText | Hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea normally catalyzes oxidation of NH(2)OH to NO(2)(-). This paper reports experiments in which HAO was thermodynamically poised to catalyze reduction of NO(2)(-) to NH(4)(+). HAO was found to catalyze the reduction of NO(2)(-) by methyl viologen monocation radical (MV(red)), displaying a hyperbolic dependence on NO(2)(-) 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 NH(2)OH by MV(red), with a hyperbolic dependence on NH(2)OH 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 MV(red) per second). We had previously demonstrated that HAO catalyzes the reduction of NO by MV(red) to yield first NH(2)OH and then NH(4)(+). Thus, overall, HAO is seen to act like a cytochrome c nitrite reductase, which catalyzes the six-electron reduction of NO(2)(-) to NH(4)(+) by MV(red). In the presence of Ru(NH(3))(2+) (Ru(II)) and Ru(NH(3))(3+) (Ru(III)) at ratios exceeding 200:1, HAO exhibited no detectable Ru(II)-NO(2)(-) oxidoreductase activity, though such activity is thermodynamically favored. On the other hand, HAO could still catalyze the oxidation of NH(2)OH to NO by Ru(III) under these conditions. The oxidative process exhibited a hyperbolic dependence on NH(2)OH concentration, with a k(cat3) of 98 ± 3 s(-1) and a K(m3) of 5.2 ± 0.8 ?M. Finally, HAO was found not to catalyze the disproportionation of NH(2)OH, 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. | lld:pubmed |
