In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper

Abstract

The effect of copper on the in vivo and in vitro activity of ammonia monooxygenase (AMO) from the nitrifying bacterium Nitrosomonas europaea was investigated. The addition of CuCl2 to cell extracts resulted in 5-to 15-fold stimulation of ammonia-dependent 02 consumption, ammonia-dependent nitrite production, and hydrazine-dependent ethane oxidation. AMO activity was further stimulated in vitro by the presence of stabilizing agents, including serum albumins, spermine, or MgCl2. In contrast, the addition of CuCl2 and stabilizing agents to whole-cell suspensions did not result in any stimulation of AMO activity. The use of the AMO-specific suicide substrate acetylene revealed two populations of AMO in cell extracts. The low, copper-independent (residual) AMO activity was completely inactivated by acetylene in the absence of exogenously added copper. In contrast, the copper-dependent (activable) AMO activity was protected against acetylene inactivation in the absence of copper. However, in the presence of copper both populations of AMO were inactivated by acetylene. ['4Clacetylene labelling of the 27-kDa polypeptide of AMO revealed the same extent of label incorporation in both whole cells and optimally copper-stimulated cell extracts. In the absence of copper, the label incorporation in cell extracts was proportional to the level of residual AMO activity. Other metal ions tested, including Zn2+, Co2+, Ni2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Cr3+, and Ag+, were ineffective at stimulating AMO activity or facilitating the incorporation of 14C label from [14C]acetylene into the 27-kDa polypeptide. On the basis of these results, we propose that loss of AMO activity upon lysis of N.europaea results from the loss of copper from AMO, generating a catalytically inactive, yet stable and activable, form of the enzyme.The chemolithoautotrophic ammonia-oxidizing bacterium Nitrosomonas europaea obtains all of its energy for growth from the oxidation of ammonia to nitrite (55). The initial oxidation of ammonia, which yields hydroxylamine as the product, is a reductant-and 02-dependent reaction catalyzed by ammonia monooxygenase (AMO):NH3 + 02 + 2e-+ 2H+ --NH20H + H20(1)Hydroxylamine is further oxidized to nitrite by hydroxylamine oxidoreductase (HAO):Two of the four electrons generated from hydroxylamine oxidation are used to support the oxidation of additional ammonia molecules, while the other two electrons enter the electron transfer chain and are used to support CO2 reduction and ATP biosynthesis (55). Despite the key role AMO plays in initiating biological nitrification, the enzyme has not been purified, and consequently, little is known about its structure, enzymatic mechanism, or the identity and function of its cofactors. This can be largely attributed to the difficulty of working with and assaying the enzyme in cell-free systems. The most convenient assays for measuring AMO activity are to monitor ammonia-dependent 02 consumption or nitrite production.These assays require a high degree of coupling: the hydroxylamine forme...

Characterization of the ferrioxamine uptake system of Nitrosomonas europaea

“…N. europaea (ATCC 19178) was cultured as described, with minor modifications (Ensign et al, 1993;Stein & Arp, 1998). The standard (Fe-replete) medium contained 10 mM Fe 3+ (FeCl 3 ) complexed with EDTA to prevent Fe from precipitation.…”

Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission

“…Nitrification of NH 3 to NO − 2 occurs in two steps (7,8). The first step is catalyzed by NH 3 monooxygenase, which uses copper (Cu) and dioxygen (O 2 ) to hydroxylate NH 3 to hydroxylamine (NH 2 OH) (9). In AOB, the second step is thought to be the four-electron oxidation of NH 2 OH to NO − 2 by NH 2 OH oxidoreductase (HAO).…”

Genera-Specific Immunofluorescence Labeling of Ammonia Oxidizers with Polyclonal Antibodies Recognizing Both Subunits of the Ammonia Monooxygenase

“…Therefore, it is assumed that their key enzyme, the ammonia monooxygenase (AMO), is highly conserved. However, little is known about the structure and function of AMO since the enzyme has not been purified [14,44,45]. Inactivation experiments suggested that the proteins AmoA and AmoB were possible subunits of AMO [33].…”