THE OXYGEN SENSOR PROTEIN, FIXL, OF RHIZOBIUM-MELILOTI - ROLE OF HISTIDINE-RESIDUES IN HEME-BINDING, PHOSPHORYLATION, AND SIGNAL-TRANSDUCTION

The two component system sensor/response regulator pair, FixL/FixJ, co ntrols the expression of Rhizobium meliloti nitrogen fixation (nif and fix) genes in response to changes in oxygen concentration. A truncate d version of FixL, FixL(), is an oxygen-binding hemoprotein kinase th at phosphorylates and dephosphorylates the nif and fix gene transcript ional activator, FixJ. Phosphorylation of FixJ is required for optimal transcriptional activation, and anaerobic conditions in vitro result in a substantial increase in the level of FixJ-phosphate. In this stud y, site-directed mutagenesis was carried out at histidine residues in FixL(). Mutant FixL(*) derivatives were purified and analyzed in vitr o for their heme/oxygen binding properties and phosphorylation/dephosp horylation activities. Mutation of histidine 285, the putative autopho sphorylation site, to glutamine results in the loss of FixL() phospho rylation activities. However, this mutant protein retains a substantia l level of FixJ-phosphate dephosphorylation activity. Mutation of hist idine 194 to asparagine results in the loss of heme binding and in the failure of FixL() to regulate its phosphorylation/dephosphorylation activities in response to changes in oxygen concentration. The FixL() H194N mutant protein also exhibits an increased FixJ phosphorylation a ctivity under aerobic conditions. This study provides further evidence for the importance of the heme binding domain of FixL() in regulatin g FixJ phosphorylation and dephosphorylation activities in response to oxygen.