O2 Protection of Nitrogenase in Frankia Isolate HFPArI3

Abstract

O2 protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (ArI3) was studied in vivo. Acetylene reduction (nitrogenase activity) was correlated with vesicle differentiation following removal of NH4+. Vesicles were broken off from the point of attachment to vegetative filaments by brief sonication. Loss of acetylene reduction was correlated with the increase in detached vesicles. Respiratory protection was suggested by the observation that O2 consumption rates in actively-fixing vesicle-containing cells were 5 times greater than rates in ammonia-grown undifferentiated cells. Furthermore, carbon limitation resulted in a decrease in the optimum O2 tension for nitrogenase activity. In actively-fixing (10 nMol C2H4/min/mg protein), aerobically induced cells, maximum acetylene reduction occurred near 20k PaO2. Acetylene reduction and O2 uptake decreased more than 10-fold in cells that were washed and incubated aerobically without a carbon source. Residual acetylene reduction activity was optimal at 5k PaO2 and nearly eliminated at 20 k PaO2. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo enzyme rates. O2 uptake rates of NH4+-grown cells showed an apparent KmO2 of approximately 1.0 um O2. In N2-fixing cultures, a KmO2 of approximately 160 um O2 was observed. Thus, respiration remains unsaturated by O2 at air saturation levels. In vivo the apparent Km for acetylene was 10-fold greater than reported in vitro values. These data show diffusion-limited kinetics and are interpreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of ArI3.