Effect of chloride on oxidation of hydroxylamine by Nitrosomonas europaea cells.

The chemoautotrophic bacterium, Nitrosomonas europaea, derives its energy from the oxidation of ammonia to nitrite. Several investigators have noted that the ability to oxidize ammonia is entirely lost upon rupture of the Nitrosomonas cell, and that, although cell-free preparations retain a small fraction of the hydroxylamine-oxidizing activity, only 30 to 70% of the total hydroxylamine that is utilized is converted to the expected physiological product, nitrite (D. J. D. Nicholas and 0. T. G. Jones, Nature 165:512, 1960; A. B. Falcone, A. L. Shug, and D. J. D. Nicholas, Biochim. Biophys. Acta 77:199, 1963; M. K. Rees, in press). The experiments presented below demonstrate that much the same alteration of hydroxylamine metabolism occurs when intact Nitrosomonas cells oxidize hydroxylamine in the presence of chloride. Pure cultures of Nitrosomonas europaea were grown and harvested as previously described (M. K. Rees and A. Nason, Biochem. Biophys. Res. Commun., 21:248, 1965). The collected cells were washed repeatedly at 4 C in 0.1 M phosphate buffer, pH 7.8, until all endogeneous nitrite was removed. To minimize the sedimentation of cellular debris in the event that lysis occurred during the washing procedure, the cells were ultimately collected by low-speed centrifugation (1,000 x g for 10 min) and were gently resuspended in the phosphate buffer at 0 C by swirling. Low-speed centrifugation was repeated each time the cells were assayed. Nitrite and hydroxylamine were assayed in triplicate essentially as described by Anderson (Biochem. J. 91:8, 1964). The simultaneous estimation of hydroxylamine and nitrite was accomplished by withdrawing a 0.5-ml portion of the reaction mixture and determining the nitrite concentration on 0.2 ml (final volume, 2 ml) and the hydroxylamine concentration on 0.3 ml (final volume, 7.5 ml). Samples containing heattreated cells (5 min at 100 C), as well as endogen ous controls, were included to correct for nonenzymatic losses of hydroxylamine and for production of nitrite. Ammonia was supplied as (NH4)2SO4 and hydroxylamine as NH20HHCl, neutralized with NaOH immediately prior to use. All chemicals were reagent grade and were used without further purification. The effect of potassium chloride on the oxidation of ammonia and hydroxylamine by intact Nitrosomonas cells is summarized in Fig. 1. For this study, a dilution of the cell suspension was determined that caused the complete utilization of either 10-4 M NH3 or 10-4 M NH20H in 2 hr at 25 C. (To obtain comparable rates, it was necessary to dilute the cells with an equal volume of buffer when hydroxylamine, rather than ammonia, served as the substrate.) Appropriately diluted cells were preincubated at 0 C in either the phosphate buffer alone or in phosphate buffer containing 0.1 M KCI. At the end of 1 hr, the cultures were warmed to 25 C, substrate (also containing 0.1 M KCI when indicated) was added, and the formation of nitrite was followed as a function of time, Figure 1 demonstrates that the addition of 0.1 M KCI had no effect on nitrite formation when the cells oxidized ammonia, and that the expected quantity of nitrite was formed when cells oxidized hydroxylamine in the absence of chloride. However, in the presence of both hydroxylamine and potassium chloride, the rate of nitrite formation, as well as the final concentration of nitrite produced, was depressed (80% of the control value). Extending the incubation time did not result in a further increase in the concentration of nitrite. The observed inhibition of nitrite formation seems to be an anion-specific effect, for, when the experiment of Fig. 1 was repeated substituting 0.1 M NaCl for KCI, the identical result was obtained. Moreover, neither a phosphate anion (0.5 M K2HPO4-KH2PO4) nor a fluoride anion (0.1 M NaFl) had an inhibitory effect on the