In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper.
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 O2 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. [14C]acetylene 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.
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Laboratory for Nitrogen Fixation Research, Oregon State University, Corvallis 97331-2902.
Hyman
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MR
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D000114:Acetylene / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism*; D002474:Cell-Free System; D003300:Copper / Q000494:pharmacology*; D004789:Enzyme Activation; D004795:Enzyme Stability / Q000187:drug effects; D004980:Ethane / Q000378:metabolism*; D006834:Hydrazines / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D009573:Nitrites / Q000378:metabolism; D009606:Nitrosomonas / Q000201:enzymology*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010105:Oxygenases / Q000187:drug effects / Q000378:metabolism*; D012709:Serum Albumin / Q000494:pharmacology
D006834:Hydrazines; D008670:Metals; D009573:Nitrites; D012709:Serum Albumin; C029424:hydrazine; D000641:Ammonia; D003300:Copper; D010105:Oxygenases; D004980:Ethane; D000114:Acetylene; C029892:cupric chloride