ABSTRACT
Conformational changes associated to sensing mechanisms of heme-based protein sensors are a key molecular event that seems to modulate not only the protein activity but also the potential of the FeIII/II redox couple of the heme domain. In this work, midpoint potentials (Em) assigned to the FeIII/II redox couple of the heme domain of FixL from Rhizobium etli (ReFixL) in the unliganded and liganded states were determined by spectroelectrochemistry in the presence of inorganic mediators. In comparison to the unliganded ReFixL protein (+19mV), the binding to ligands that switch off the kinase activity induces a negative shift, i. e. Em=-51, -57 and -156mV for O2, imidazole and CN-, respectively. Upon binding to CO, which does not affect the kinase active, Em was observed at +21mV. The potential values observed for FeIII/II of the heme domain of ReFixL upon binding to CO and O2 do not follow the expected trend based on thermodynamics, assuming that positive potential shift would be expected for ligands that bind to and therefore stabilize the FeII state. Our results suggest that the conformational changes that switch off kinase activity upon O2 binding have knock-on effects to the local environment of the heme, such as solvent rearrangement, destabilize the FeII state and counterbalances the FeII-stabilizing influence of the O2 ligand.
