The two-component system sensor/response regulator pair, FixL/FixJ, controls the expression of Rhizobium meliloti nitrogen fixation (nif and fix) genes in response to changes in oxygen concentration. A truncated version of FixL, FixL*, is an oxygen-binding hemoprotein kinase that phosphorylates and dephosphorylates the nif and fix gene transcriptional 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 study, site-directed mutagenesis was carried out at histidine residues in FixL*. Mutant FixL* derivatives were purified and analyzed in vitro for their heme/oxygen binding properties and phosphorylation/dephosphorylation activities. Mutation of histidine 285, the putative autophosphorylation site, to glutamine results in the loss of FixL* phosphorylation activities. However, this mutant protein retains a substantial level of FixJ-phosphate dephosphorylation activity. Mutation of histidine 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 activity under aerobic conditions. This study provides further evidence for the importance of the heme binding domain of FixL* in regulating FixJ phosphorylation and dephosphorylation activities in response to oxygen.