Estudo espectroeletroquímico da hemeproteína sensora Rhizobium etli FixL

• Ano de defesa: 2017
• Autor(a) principal: Felício, Nathalie Honorio
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Bioeletroquímica; Proteína FixL; Espectroeletroquímica; Hemeproteína; Sistema de dois componentes
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/59248

This work presents the expression, purification and spectroelectrochemistry results obtained for the sensor hemeprotein FixL from Rhizobium etli (ReFixL). In addition, the spectroelectrochemistry data allowed the correlation between the potential values of the redox reaction of the Fe metal center presented in the heme group with the kinase activity of the ReFixL protein. The ReFixL protein, expressed and purified in its met-ReFixL(FeIII) form, showed molecular mass value of about 70 kDa (ReFixL). The values of the redox potentials of the studied protein was determined by spectroelectrochemical titration in buffer solution (pH 8 or 9.5) containing redox mediators that allowed to cover a potential range from 500 to + 500 mV vs ENH. Such measurements were performed for the proteins free of coordination and bonded (coordination to the Fe atom of the heme group) to oxygen (O2), carbon monoxide (CO), cyanide (CN) and imidazole molecules; all of them relevant in the activation/inactivation kinase activity in the case of ReFixL. The spectroscopic profiles of the ReFixL protein supported these species were bound to CO and O2 in the reduced state and to imidazole and CN in the oxidized state, being observed, respectively, the following potential values: +21, 51, 57 and 156 mV vs ENH. The inactivation of the kinase activity of the ReFixL protein is observed upon binding to O2, imidazole and CN− molecules, whose redox potentials show negative values. The negative potential shift in relation to the reduced non-bonded state of the protein (deoxy-ReFixL; +19 mV vs ENH) indicates the reduction potential is more affected by conformational changes induced by the ligands than the electron density donor/acceptor character of the ligands do. The potential values observed for Fe-O2 do not follow the expected based on its thermodynamics, assuming the expected potential shift would have been positive for ligands that bind to reduced state FeII. Our results suggest that conformational changes that follow due to kinase activity switching off upon O2 binding have some knock-on effect on the heme environment, that destabilize the FeII state and conterbalances the FeII stabilization normally caused by a ligand to the reduced form.