A soluble form of ammonia monooxygenase in Nitrosomonas europaea.
Ammonia monooxygenase (AMO) of Nitrosomonas europaea is a metalloenzyme that catalyzes the oxidation of ammonia to hydroxylamine. This study shows that AMO resides in the cytoplasm of the bacteria in addition to its location in the membrane and is distributed approximately equally in both subcellular fractions. AMO in both fractions catalyzes the oxidation of ammonia and binds [(14)C]acetylene, a mechanism-based inhibitor which specifically interacts with catalytically active AMO. Soluble AMO was purified 12-fold to electrophoretic homogeneity with a yield of 8%. AMO has a molecular mass of approximately 283 kDa with subunits of ca. 27 kDa (alpha-subunit, AmoA), ca. 42 kDa (beta-subunit, AmoB), and ca. 24 kDa (gamma-subunit, cytochrome c(1)) in an alpha(3)beta(3)gamma(3) sub-unit structure. Different from the beta-subunit of membrane-bound AMO, AmoB of soluble AMO possesses an N-terminal signal sequence. AMO contains Cu (9.4+/-0.6 mol per mol AMO), Fe (3.9+/-0.3 mol per mol AMO), and Zn (0.5 to 2.6 mol per mol AMO). Upon reduction the visible absorption spectrum of AMO reveals absorption bands characteristic of cytochrome c. Electron para-magnetic resonance spectroscopy of air-oxidized AMO at 50 K shows a paramagnetic signal originating from Cu(2+) and at 10 K a paramagnetic signal characteristic of heme-Fe.
Gilch
Stefan
S
Department of Microbiology, University of Bayreuth, Bayreuth, Germany.
Meyer
Ortwin
O
Schmidt
Ingo
I
D000114:Acetylene / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004586:Electrophoresis; D043370:Nitrosomonas europaea / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012995:Solubility
D001426:Bacterial Proteins; D045304:Cytochromes c; D010088:Oxidoreductases; C045582:ammonia monooxygenase; D000114:Acetylene