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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Ludwig, Stephan / Sies, Helmut / Turk, Boris / Wittinghofer, Alfred

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A soluble form of ammonia monooxygenase in Nitrosomonas europaea

Stefan Gilch1 / Ortwin Meyer1 / Ingo Schmidt1

1Department of Microbiology, University of Bayreuth, D-95447 Bayreuth, Germany

Corresponding author

Citation Information: Biological Chemistry. Volume 390, Issue 9, Pages 863–873, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: 10.1515/BC.2009.085, May 2009

Publication History

Received:
2009-02-17
Accepted:
2009-04-25
Published Online:
2009-05-20

Abstract

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 [14C]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 (α-subunit, AmoA), ca. 42 kDa (β-subunit, AmoB), and ca. 24 kDa (γ-subunit, cytochrome c 1) in an α3β3γ3 sub-unit structure. Different from the β-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 Cu2+ and at 10 K a paramagnetic signal characteristic of heme-Fe.

Keywords: acetylene; ammonia monooxygenase (AMO); AmoA; AmoB; copper and iron enzyme; cytochrome c1; N-terminal signal sequence; purification

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