KINETIC-STUDIES OF AMMONIA MONOOXYGENASE INHIBITION IN NITROSOMONAS-EUROPAEA BY HYDROCARBONS AND HALOGENATED HYDROCARBONS IN AN OPTIMIZED WHOLE-CELL ASSAY

The inhibitory effects of 15 hydrocarbons and halogenated hydrocarbons on NH, oxidation by ammonia monooxygenase (AMO) in intact cells of the nitrifying bacterium Nitrosomonas europaea were determined. Determination of AMO activity, measured as NO2- production, required coupling of hydroxylamine oxidoreductase (HAO) activity with NH3-dependent NH2OH production by AMO. Hydrazine, an alternate substrate for HAO, was added to the reaction mixtures as a source of reductant for AMO. Most inhibitors exhibited competitive or noncompetitive inhibition patterns. The competitive character generally decreased (K(iE)/K(iES) increased) as the molecular size of the inhibitors increased. For example, CH4 and C2H4 were competitive inhibitors of NH3 oxidation, whereas the remaining alkanes (UP to C4) and monohalogenated (Cl, Br, I) alkanes were noncompetitive. Oxidation of C2H5Br (noncompetitive) increased as the NH4+ concentration increased up to 40 mM, whereas oxidations of inhibitors with competitive character (K(iE) much less than K(iES)) were diminished at 40 mM NH4+. Multichlorinated compounds produced nonlinear Lineweaver-Burk plots. Iodinated alkanes (CH3I, C2H5I) and C2Cl4 were potent inhibitors of NH3 oxidation. Maximum rates of NH3, C2H4, and C2H6 oxidations were approximately equivalent, suggesting a common rate-determining step. These data support an active-site model for AMO consisting of an NH3-binding site and a second site that binds noncompetitive inhibitors, with oxidation occurring at either site.