Regulation of the kinase activity of heme protein FixL from the two-component system FixL/FixJ of Rhizobium meliloti

The Rhizobium meliloti two-component system FixL/FixJ regulates nitrogen fixation in response to oxygen during symbiosis. Fix J is a transcriptional activator of critical nif and fix promoters; its in vivo activity is enhanced by FixL in diminished oxygen (David, M., Daveran, M.-L., Batut, J., Dedieu, A., Domergue, O., Ghai, J., Hertig, C., Boistard, P., and Kahn, D. (1988) Cell 54, 671-683; Virts, E. L., Stanfield, S. W., Helinski, D. R., and Ditta, G. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3062-3065). FixL* is a soluble truncated version of FixL that contains heme; it catalyzes its autophosphorylation and the phosphorylation of FixJ (Gilles-Gonzalez, M. A., Ditta, G. S., and Helinski, D. R. (1991) Nature 350, 170-172). We examine the kinetics of phosphoryl transfer in this system. First, there is a slow autophosphorylation of FixL* in ATP that is accelerated in the absence of oxygen and in the presence of Mn²⁺. This reaction is reversible, i.e. phospho-FixL* reacts with ADP to generate ATP. Since the reverse reaction is faster, most FixL* is not phosphorylated at equilibrium. Next, there is a rapid phosphoryl transfer directly from phospho-FixL* to FixJ that is unaffected by oxygen. Finally, phospho-FixJ is hydrolyzed; this reaction is very fast and not controlled by oxygen. We propose that in addition to the oxygen signal previously noted in vivo, energy charge and manganese concentration are also indicators of symbiosis that impact on the induction of nitrogen fixation genes.