title: Electron paramagnetic studies of the copper and iron containing soluble ammonia monooxygenase from Nitrosomonas europaea.
abstract: Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g(parallel) = 2.24, A(parallel) = 18.4 mT and g(perpendicular) = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g(parallel) = 2.29, A(parallel) = 16.1 mT and g(perpendicular) = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu(2+) with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu(2+) and three diamagnetic Cu(1+) per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per alphabetagamma-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe(3+) was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu(2+)-EPR signal.
authors.affiliation: Lehrstuhl für Mikrobiologie, Universität Bayreuth, Bayreuth 95447, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Gilch
authors.forename: Ortwin
authors.initials: O
authors.lastname: Meyer
authors.forename: Ingo
authors.initials: I
authors.lastname: Schmidt
mesh_terms: D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D007501:Iron / Q000737:chemistry*; D043370:Nitrosomonas europaea / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003300:Copper; D007501:Iron; D010088:Oxidoreductases; C045582:ammonia monooxygenase