Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids

The relationship between the O-2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP acid ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O-2 coincided with an increase of the cellular ATP/ADP ratio, When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity, To explain these observations, the effects of nitrogenase activity and CCCP on the 0, consumption rate of R. leguminosarum bacteroids were determined, From 100 to 5 mu M O-2, a decline in the O-2 consumption rate was observed to 50 to 70% of the maximal O-2 consumption rate, A determination of the redox state of the cytochromes during an O-2 consumption experiment indicated that at O-2 concentrations above 5 mu M, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O-2 concentrations of >300 mM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (K-m of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O-2.