title: Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47.
abstract: Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c - namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death - is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), Centro de Investigaciones Científicas Isla de la Cartuja (cicCartuja), Universidad de Sevilla, Consejo Superior de Investigaciones Científicas (CSIC), Avda. Américo Vespucio 49, Sevilla 41092, Spain.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Guerra-Castellano
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), Centro de Investigaciones Científicas Isla de la Cartuja (cicCartuja), Universidad de Sevilla, Consejo Superior de Investigaciones Científicas (CSIC), Avda. Américo Vespucio 49, Sevilla 41092, Spain. Electronic address: idiazmoreno@us.es.
authors.forename: Irene
authors.initials: I
authors.lastname: Díaz-Moreno
authors.affiliation: Institute of Biocomputation and Physics of Complex Systems (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Mariano Esquillor s/n, 50018 Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain; Fundacion ARAID, Government of Aragon, Maria de Luna 11, 50018 Zaragoza, Spain.
authors.forename: Adrián
authors.initials: A
authors.lastname: Velázquez-Campoy
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), Centro de Investigaciones Científicas Isla de la Cartuja (cicCartuja), Universidad de Sevilla, Consejo Superior de Investigaciones Científicas (CSIC), Avda. Américo Vespucio 49, Sevilla 41092, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: De la Rosa
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), Centro de Investigaciones Científicas Isla de la Cartuja (cicCartuja), Universidad de Sevilla, Consejo Superior de Investigaciones Científicas (CSIC), Avda. Américo Vespucio 49, Sevilla 41092, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Díaz-Quintana
mesh_terms: D002308:Cardiolipins / Q000737:chemistry; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry* / Q000502:physiology; D004579:Electron Transport; D004795:Enzyme Stability; D009154:Mutation; D012694:Serine; D013912:Threonine
chemical_list: D002308:Cardiolipins; D013912:Threonine; D012694:Serine; D045304:Cytochromes c; D020169:Caspases
keywords: Caspase activity; Cytochrome c; Electron transport chain; Liposomes binding; Peroxidase activity; Phosphorylation