Photoacoustic Calorimetry Studies of O 2 -Sensing FixL and (R200, I209) Variants from Sinorhizobium meliloti Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain.

FixL is an oxygen-sensing heme-PAS protein that regulates nitrogen fixation in the root nodules of plants. In this paper, we present the first photothermal studies of the full-length wild-type FixL protein from Sinorhizobium meliloti and the first thermodynamic profile of a full-length heme-PAS protein. Photoacoustic calorimetry studies reveal a quadriphasic relaxation for Sm FixL*WT and the five variant proteins ( Sm FixL*R200H, Sm FixL*R200Q, Sm FixL*R200E, Sm FixL*R200A, and Sm FixL*I209M) with four intermediates from <20 ns to ∼1.5 μs associated with the photodissociation of CO from the heme. The altered thermodynamic profiles of the full-length Sm FixL* variant proteins confirm that the conserved heme domain residues R200 and I209 are important for signal transduction. In contrast, the truncated heme domain, Sm FixLH128-264 , shows only a single, fast monophasic relaxation at <50 ns associated with the fast disruption of a salt bridge and release of CO to the solvent, suggesting that the full-length protein is necessary to observe the conformational changes that propagate the signal from the heme domain to the kinase domain.