Energy-conserving reactions in phosphorylating electron-transport particles from Nitrobacter winogradskyi. Activation of nitrite oxidation by the electrical component of the protonmotive force

Abstract

1. In electron-transport particles (ET particles) prepared from Nitrobacter winogradskyi, the uncoupling agent carbonyl cyanide phenylhydrazone increased the rate of NADH oxidation but decreased the rate of oxidation of NO2-. Its effectiveness in stimulating NADH oxidation closely paralleled its effectiveness in inhibiting NO2- oxidation. 2. In the presence of ADP and phosphate the oxidation of NADH was stimulated, whereas the oxidation of NO2- was inhibited. In the presence of excess of Pi the concentration dependence with respect to ADP was the same for acceleration of NADH oxidation and inhibition of NO2- oxidation. 3. Oligomycin inhibited NADH oxidation and stimulated the oxidation of NO2-. The concentration of oligomycin required to produce half-maximal effect in both systems was the same. 4. The apparent Km for NO2- was not affected by ADP together with Pi, by uncoupling agent or by oligomycin. 5. With NADH as substrate, classical respiratory control was observed. With NO2- as substrate the respiratory-control ratio was less than unity. 6. A reversible uptake of H+ accompanied the oxidation of NO2- by ET particles. 7. In the presence of NH4Cl or cyclohexylamine hydrochloride, H+ uptake was abolished and increased rates of NO2- oxidation were observed. When valinomycin was present in the reaction medium, low concentrations of NH4Cl inhibited NO2- oxidation. 8. Pretreatment of ET particles with oligomycin enhanced the stimulation of NO2- oxidation induced by NH4Cl or by cyclohexylamine hydrochloride. Pretreatment with the uncoupler carbonyl cyanide phenylhydrazone prevented these stimulations. 9. In the presence of dianemycin together with K+, the uptake of H+ was abolished and the rate of NO2- oxidation was increased. In contrast, in the presence of valinomycin together with K+, the uptake of H+ was increased, and the rate of NO2- oxidation decreased. 10. Sodium tetraphenylboron was found to be an inhibitor of NO2- oxidation, but caused a stimulation of NADH oxidation which was dependent on the presence of NH4Cl or cyclohexylamine hydrochloride. 11. It is concluded that the enhanced rate of NO2- oxidation observed in the absence of energy-dissipating processes clearly relates to some state before the involvement of adenine nucleotides, and it is suggested that the oxidation of NO2- generates a protonmotive force, the electrical component of which controls the rate of NO2- oxidation.