The Oxygen Sensitivity of Nitrogenase in Rhodobacter capsulatus: Repression and Inactivation

Abstract

Abstract In chemostat cultures of Rhodobacter capsulatus, growing aerobically in darkness, in situ nitrogen fixation occurred at significantly lower oxygen concentrations than the acetylene reduction activity of nitrogenase, as determined with samples from the culture under opti­ mum assay conditions. In contrast to nitrogenase of cultures growing diazotrophically at low oxygen, nitrogenase present in inactive form at the higher oxygen concentrations could not be activated to fix nitrogen by increasing i.) the energy supply by illuminating the cultures, ii.) the supply of cells with electron donor and iii.) cellular respiration. These results suggest that oxygen controls cellular nitrogen fixation directly rather than indirectly by interfering with the general metabolism. On this basis, the sensitivity toward oxygen of nif gene expression as well as of nitrogenase polypeptide accumulation was studied. The nifH promoter was active up to about 40% air saturation, exhibiting a biphasic sensitivity to oxygen, i.e. steep decreases in activity between 1 to 2% and between 10 to about 20% air saturation. A similar behaviour was observed with respect to cellular levels of both polypeptides of component 1 (Rc1) of nitrogenase, which were accumulated up to about 20% air saturation. Component 2 (Rc2) was less oxygen-sensitive than Rc1, its steady state level reached a maximum at 2% air saturation. A sudden increase in the ambient oxygen concentration, i. e. oxygen stress, revealed that, in vivo, nitrogenase was fairly stable against oxygen damage. Modification of Rc2 by ADP-ribosylation occurred under ammonium shock conditions, regardless of whether the cells fixed nitrogen or not. But it was not observed with cultures growing aerobically in the dark at steady state or under oxygen stress. Results obtained with mutant W107I of R. capsulatus lacking the modifying/demodifying enzymes supported the conclusion that modifi­ cation of Rc2 was not required for the inactivation of nitrogenase by oxygen.