Ammonia monooxygenase of Nitrosomonas europaea: Structure, function and catalytic mechanism
Zusammenfassung der Projektergebnisse
For the first time a soluble functional AMO was described in N. europaea in addition to the membrane-bound enzyme. Soluble and membrane-bound AMO occur in almost the same amounts in vivo and both conformations are catalytically active. Soluble AMO has a molecular mass of about 316 kDa and is a Cu-, Fe- (heme and non-heme Fe), and probably Zn-containing enzyme composed of the subunits AmoA (~27 kDa a-subunit), AmoB (~42 kDa ß-subunit), and cytochrome c1 (~24 kDa y-subunit) with an a3ß3y3 subunit structure. AMO contains 9.4 ± 0.6 mol Cu (mol AMO)-1, 3.9 ±0.3 mol Fe (mol AMO)-1, and 0.5 to 2.6 mol Zn (mol AMO)-1. Upon reduction the visible absorption spectrum of AMO reveals absorption bands characteristic of cytochrome c. Electron paramagnetic resonance spectroscopy of air-oxidized AMO at 50 K shows a paramagnetic signal originating from Cu2+ and at 10 K a paramagnetic signal characteristic of heme-Fe. The observation that a 25 amino acid signal peptide is cleaved from the membrane-bound form of the enzyme, but not from the soluble AMO lead to the assumption that this N-terminal sequence alters folding of AMO resuhing in a soluble and a membrane-bound conformation. The specific mechanismbased inactivator acetylene is oxidized by AMO to ketene which binds covalently at His 191 and upon ketene binding about three Cu-atoms are liberated from the enzyme. Apparently the combination of ketene binding and Cu liberation irreversible inactivates AMO. The results give evidence that His 191 contributes to the coordination of an active Cu containing catalytic center responsible for ammonia oxidation. In actively ammonia oxidizing N. europaea the periplasmic space is acidified by hydroxylamine oxidation and the set-up of a PMF. The pH value range between 5 and 6 and both soluble and membrane-bound AMO are inactive at such a low pH value. As a consequence, ammonia oxidation catalyzed by both AMO's must occur in the cytoplasmic space where the pH value between 7 and 8 is optimal for ammonia oxidation by AMO. From an energetic point of view the oxidation of ammonia in the cytoplasmic space is advantageous, because the reaction consumes 2 H+/NH3 which raises the proton gradient (PMF), whereas an oxidation in the periplasmic space would diminish the proton gradient. Soluble AMO is able to catalyze a disproportion of hydroxylamine to ammonia, nitrate, and nitrite according to the equation 3.2 NH2OH -> 2 NH3 + 0.8 NO3- + 0,4 NO2- + 3.6 H+ + 2.4 e- The reaction provides reducing equivalents, but in contrast to ammonia oxidation the efficiency is low and a significant growth of N. europaea on hydroxylamine was not observed so far. Hydroxylamine is an intermediate of the sequential oxidation of ammonia by AMO and HAO and unbalanced enzyme activities can result in an overproduction and accumulation of hydroxylamine. Hydroxylamine disproportion would be able to counteract an accumulation of hydroxylamine and to protect N. europaea from its toxic effect. The physical separation of ammonia and hydroxylamine oxidation (cytoplasmic/periplasmic) might complicate the synchronization of both reactions and hydroxylamine disproportion by AMO might have its function as a mechanism to detoxify hydroxylamine and to partially regenerate the substrate ammonia. In this perspective, hydroxylamine disproportion contributes to a regulation of inorganic N-metabolism and energy conservation in N. europaea.
Projektbezogene Publikationen (Auswahl)
-
(2009) Interaction of the mechanism-based inactivator acetylene with ammonia monooxygenase of Nitrosomonas europaea. Microbiology 155,279-284
Gilch S., Vogel M., Lorenz MW., Meyer O. & Schmidt I.
