A soluble form of ammonia monooxygenase in Nitrosomonas europaea

Ammonia monooxygenase (AMO) ofNitrosomonas europaeais 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, cytochromec1) in an α3β3γ3sub-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 cytochromec. 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.

Trichloroethylene metabolism in vitro: an EPR/SPIN trapping study

Trichloroethylene (TCE) was hypothesized to produce free radicals which could be detected using electron para magnetic resonance spectroscopy with the spin trap, PBN (α-phenyl tert-butyl nitrone). The free radicals detected following incubation of precision cut liver slices in media containing 10 mM PBN had hyperfine coupling constants aN=1.61 mT and aH=0.325 mT. There was a linear increase in free radicals detected in the bathing media when the headspace TCE concentration was increased from 2500- 10 000 p.p.m. The levels of conjugated dienes measured in the slices incubated in PBN supplemented media were less than slices exposed to TCE in incubation media without PBN. The PBN trap may act as a scavenger preventing the propagation of free radicals and inhibiting lipid peroxida tion. The experiments suggest that free radical formation by TCE leads to a concomitant increase in conjugated dienes in liver slices which may contribute to the pathological changes which occur in liver following TCE exposure.