RESPIRATORY CONTROL DETERMINES RESPIRATION AND NITROGENASE ACTIVITY OF RHIZOBIUM-LEGUMINOSARUM BACTEROIDS

The relationship between the O-2 input rate into a suspension of Rhizo bium leguminosarum bacteroids, the cellular ATP acid ADP pools, and th e whole-cell nitrogenase activity during L-malate oxidation has been s tudied. It was observed that inhibition of nitrogenase by excess O-2 c oincided with an increase of the cellular ATP/ADP ratio, When under th is condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrog enase regained activity, To explain these observations, the effects of nitrogenase activity and CCCP on the 0, consumption rate of R. legumi nosarum 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 cytochr omes during an O-2 consumption experiment indicated that at O-2 concen trations above 5 mu M, electron transport to the cytochromes was rate- limiting oxidation and not the reaction of reduced cytochromes with ox ygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deox ygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respira tory control. The dehydrogenase activities of the respiratory chain we re 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. leguminosaru m bacteroids takes place via a respiratory chain terminating at a high -affinity oxidase system, (ii) the activity of the respiratory chain i s inhibited by the proton motive force, and (iii) ATP hydrolysis by ni trogenase can partly relieve the inhibition of respiration by the prot on motive force and thus stimulate respiration at nanomolar concentrat ions of O-2.