Properties of the alfalfa–Rhizobium meliloti symbiotic nitrogenase enzyme system in vivo and in vitro

Abstract

A comparison of hydrogen production and acetylene reduction by excised alfalfa nodules, nitrogenase proteins isolated from Rhizobium meliloti bacteroids, and nitrogenase proteins purified from Klebsiella pneumoniae revealed several differences in the catalytic properties of the in vivo and in vitro systems. In all cases, assays for nitrogenase activity were carried out at near-physiological enzyme concentrations. Intact nodules maintained high nitrogenase activity to much lower reaction temperatures than the isolated enzymes. The Q10 value for acetylene reduction was 1.4 for nodules, but exceeded 5 for isolated R. meliloti nitrogenase in the temperature range 12–28 °C. The reduction of substrates requiring more than two reducing equivalents by the isolated enzyme system was severely restricted by the effects of reaction temperature on electron flux, as shown by very low values for electron allocation to dinitrogen determined indirectly at 12 °C. Temperature-induced lags in the initial reaction velocity of the reconstituted Klebsiella nitrogenase system were not observed with the isolated, but unseparated, R. meliloti nitrogenase proteins when acetylene was the substrate. The respiration-supported nodule system was not saturated with oxygen under air at reaction temperatures between 12 and 25 °C. Measurements of the adenine nucleotide levels of whole nodules indicated that the observed effects of reaction temperature on nitrogenase in vitro may be partially compensated in vivo by increases in adenylate energy charge and marked decreases in the ratio of ADP/ATP which occur as the nodule temperature is lowered. From the results reported here, it is clear that the R. meliloti – alfalfa nitrogenase system is limited in activity by oxygen supply. However, electron flux is sufficient within the physiological temperature range to maintain the Mo–Fe protein in the catalytically active state.