Insights into heme-based O2 sensing from structure-function relationships in the FixL proteins.

FixL proteins are bacterial heme-containing signal transduction proteins responsible for sensing the O(2) concentration in the organism's environment. In Sinorhizobium meliloti FixL is a protein histidine kinase that, together with its response regulator FixJ, constitute an oxygen-sensitive switch for regulation of the organism's nitrogen fixation and microaerobic respiration genes. The O(2) sensitivity of the switch is such that it transitions during the process of symbiosis in alfalfa roots. Bradyrhizobium japonicum FixL similarly regulates microaerobic and anaerobic respiration genes during symbiosis in soybean roots. FixLs responds to low oxygen concentrations with increased autophosphorylation activity of their kinase domains. The phosphorylated FixL provides a phosphoryl group to FixJ within a FixLJ complex. The phosphorylated FixJs are transcriptionally active toward their target genes. The FixL kinase domain is inhibited when the heme in FixL is oxygenated. Kinetic and thermodynamic studies of ligand binding to both ferrous and ferric FixLs have shown a generally low affinity for ligands relative to myoglobins. These relatively low ligand affinities are attributable almost completely to diminished rates of ligand binding. The heme and its environment in liganded and unliganded FixLs have been characterized by UV-visible spectroscopy, resonance Raman spectroscopy, EXAFS, and X-ray crystallography. These studies have revealed that in the purified proteins, the heme is converted from a six-coordinate low spin state to a five-coordinate high spin state upon O(2) release. Comparisons of spectroscopic and structural characteristics of deoxyFixL with oxyFixL, met-FixL-CN, FixL-CO, and FixL-NO complexes indicate that distal affects in the heme pocket are, at least in part, responsible for communicating the ligation state of the heme to the kinase domain. The mechanisms by which ligand binding events are communicated from the heme to the kinase domain involves propagation and/or amplification of the ligation-coupled conformational transitions of the heme and its immediate protein environment. More recently, time-resolved experiments examining the nonequilibrium, ligand-coupled dynamics initiated by O(2), CO, and NO photolysis from the corresponding FixL complexes have begun to shed light on the landscape of the switching coordinate. Current thinking and understanding of the mechanism for signal transduction in the FixLJ systems are discussed in the context of these physical investigations.