Ammonia monooxygenase of Nitrosomonas europaea: Structure, function and catalytic mechanism

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Nitrosomonas europaea oxidizes ammonia to nitrite under either oxic or anoxic chemolithoautotrophic conditions. Two enzymes have key functions in this energy yielding reaction: The ammonia monooxygenase (AMO), catalyzing the oxidation of ammonia to hydroxylamine, and the hydroxylamine oxidoreductase (HAO), oxidizing hydroxylamine to nitrite. The 2.8 Å structure of the hydroxylamine oxidoreductase was resolved by Igarashi et al. (1997). The objectives of this project are to purify the AMO from N. europaea, to heterologously express the AMO in E. coli and to purify the protein, to establish a protocol for the crystallization of the protein from both sources, and to solve the quaternary structure including the architecture of its catalytic center and co-factors present. Relevant distances will be verified by X-ray absorption spectroscopy, and redox states will be identified by electron paramagnetic resonance (EPR) spectroscopy. The enzyme assay will be further optimized to characterize the catalytic activity of the AMO. Our strategy to purify the enzyme under anoxic conditions in the presence of NO and copper led already to first promising results and is, in combination with the heterologous expression of the AMO proteins, the fundament for a successful implementation of this project. The ultimate goal is to combine structural and biochemical data to develop a detailed model of the catalytic function of the AMO at the molecular level.