Abstract
In the denitrifiers, Pseudomonas denitrificans and Pseudomonas aeruginosa, nitrogen oxide respirations were reversibly inhibited by several weak acid-type uncouplers of oxidative phosphorylation. Their effectiveness as uncouplers stood in about the same rank as their effectiveness as inhibitors. In contrast, uncouplers increased the rate of oxygen respiration (release of respiratory control) in these two organisms and seemed not to inhibit even at rather high concentrations. The mechanism of inhibition of nitrogen oxide respiration by un- couplers was explored and several lines of evidence ruled out an inhibition which depended on collapse of the proton motive force per se. The concentrations of uncouplers required to in- hibit nitrogen oxide respirations in Ps. denitrificans were found to be at least ten times higher than those required (a) to inhibit respiration-driven amino acid uptake, (b) to release respiratory control in the case of oxygen respiration, and (c) to increase membrane proton conductivity to such levels that endergonic reactions dependent on proton motive force must, by calcula- tion, be inhibited. Neither valinomycin/K+ (to collapse membrane potential) nor nigericin (to collapse ) had an inhibitory effect on nitrogen oxide respirations, whether present separately or together. In addition, nonuncoupling analogues of the nitrophenol and carbonyl cyanide phenylhydrazone uncouplers were effective in inhibiting respiration. The inhibitions are attributed in large part to the hydrophobic properties of these compounds. Spectrophotometric observations suggested that the site of inhibition lay on the reducing side of cytochromes b and c. In the steady states of nitrogen oxide respirations, cytochromes b and c remained nearly completely oxidized in the presence of uncouplers, and the transition from steady state to reduced state upon the exhaustion of nitrogen oxide was inhibited. Certain trivial explanations for the inhibitory effect of uncouplers, such as substrate starvation, were ruled out.