Ligand binding dynamics in heme-based oxygen sensor FixL studied by ultrafast spectroscopy

International audienceHeme-based oxygen sensors are part of ligand specific two-component regulatory systems. FixL from Bradyrhizobium japonicum is an example of such a sensor. In this protein the binding of oxygen to the heme in the receptor domain causes changes in an associated enzymatic domain that eventually regulates transcription factors. We used femtosecond absorption spectroscopy to investigate the binding dynamics of O2, CO and NO and the characteristics of the first signaling intermediate upon oxygen release with wild type and mutant forms of FixL. In wild-type specifically, the oxygen binding kinetics in FixL are extremely fast and efficient. Within 20 ps, 90% of oxygen is already recombined in a geminate way and only 10% of the dissociated oxygen leaves the heme pocket, showing that the heme pocket acts as an oxygen trap [1]. The heme spectrum after photolysis of oxygen is perturbed compared to the steady state oxygen-binding spectrum, which shows the residual interaction with the dissociated ligand. Arginine 220 is located in the heme pocket and is proposed to stabilize the bound ligand and participate in the signal transduction [2]. Substitution of R220 with isoleucine, glutamic acid and glutamate all had similar effects: It strongly decreased the part of fast oxygen geminate recombination pointing at an important role of this residue in oxygen trapping. Also the residual interaction with dissociated oxygen is lost as witnessed by the less perturbed dissociation spectrum. The steady-state heme conformation and ligand characteristics are similar in FixL, the oxygen sensor, and myoglobin, the oxygen storage protein. In the heme pocket of Mb a histidine can form a hydrogen bond to the ligand. We prepared the R220H mutant of FixL in which the oxygen binding kinetics became biphasic. In addition to the fast phase of oxygen binding appeared a slower, nanosecond component. The amplitude of each phase was about 50% of the total amplitude. The binding of CO also was dramatically affected. In the R220H mutant about 50% of carbon monoxide rebinds geminately with a time constant 2.4 ns, which is observed nor in WT FixL nor in Mb, where only bimolecular binding of CO occurs. The ensemble of our data indicate that R220 plays a key role in transmission of the signal within the heme domain. Preliminary MD simulations support this vie