The Heme-Based Oxygen Sensor Rhizobium etli FixL: Influence of Auxiliary Ligands on Heme Redox Potential and Implications on the Enzyme Activity.
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/IIredox couple of the heme domain. In this work, midpoint potentials (Em) assigned to the FeIII/IIredox 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, Emwas observed at +21mV. The potential values observed for FeIII/IIof the heme domain of ReFixL upon binding to CO and O2do 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 FeIIstate. Our results suggest that the conformational changes that switch off kinase activity upon O2binding have knock-on effects to the local environment of the heme, such as solvent rearrangement, destabilize the FeIIstate and counterbalances the FeII-stabilizing influence of the O2ligand.
Laboratório de Bioinorgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CEP 60455-760, Fortaleza, Ceará, Brazil; School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, 4072, Australia.
Nathalie
N
Honorio-Felício
Laboratório de Bioinorgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CEP 60455-760, Fortaleza, Ceará, Brazil.
Marta S P
MS
Carepo
Laboratório de Bioinorgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CEP 60455-760, Fortaleza, Ceará, Brazil.
Tércio
T
de F Paulo
Laboratório de Bioinorgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CEP 60455-760, Fortaleza, Ceará, Brazil.
Luiz Gonzaga
LG
de França Lopes
Laboratório de Bioinorgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CEP 60455-760, Fortaleza, Ceará, Brazil. Electronic address: eduardohss@dqoi.ufc.br.
Eduardo H S
EH
Sousa
Laboratório de Bioinorgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CEP 60455-760, Fortaleza, Ceará, Brazil. Electronic address: izaura@dqoi.ufc.br.
Izaura C N
IC
Diógenes
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, 4072, Australia. Electronic address: p.bernhardt@uq.edu.au.
Paul V
PV
Bernhardt
D001426:Bacterial Proteins / Q000737:chemistry*; D015374:Biosensing Techniques; D002248:Carbon Monoxide / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D000071677:Histidine Kinase; D010100:Oxygen / Q000032:analysis*; D042163:Rhizobium etli / Q000737:chemistry*
D001426:Bacterial Proteins; D006420:Hemeproteins; D006418:Heme; D002248:Carbon Monoxide; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen
FixL; Heme-based sensor; kinase activity; redox potential; spectroelectrochemistry