title: In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803.
abstract: The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane.
authors.affiliation: Estación Experimental Aula Dei (EEAD), Consejo Superior de Investigaciones Científicas (CSIC), Carretera Montañana 1005, 50059 Zaragoza, Spain.
authors.forename: María A
authors.initials: MA
authors.lastname: Luján
authors.affiliation: Instituto Ciencia de Materiales de Aragón (ICMA) (CSIC-Universidad de Zaragoza), C/Pedro Cerbuna 12, 50009 Zaragoza, Spain.
authors.forename: Jesús I
authors.initials: JI
authors.lastname: Martínez
authors.affiliation: Instituto Ciencia de Materiales de Aragón (ICMA) (CSIC-Universidad de Zaragoza), C/Pedro Cerbuna 12, 50009 Zaragoza, Spain.
authors.forename: Pablo J
authors.initials: PJ
authors.lastname: Alonso
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF) (Universidad de Sevilla-CSIC), Avda. Américo Vespucio 49, 41092 Sevilla, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Torrado
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF) (Universidad de Sevilla-CSIC), Avda. Américo Vespucio 49, 41092 Sevilla, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Roncel
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF) (Universidad de Sevilla-CSIC), Avda. Américo Vespucio 49, 41092 Sevilla, Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Ortega
authors.affiliation: Instituto de Biocomputación y Física de Sistemas Complejos - Unidad BIFI-IQFR (CSIC) (Universidad de Zaragoza), C/Mariano Esquilar s/n, 50018 Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular (Universidad de Zaragoza), Plaza San Francisco s/n, 50009 Zaragoza, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Sancho
authors.affiliation: Estación Experimental Aula Dei (EEAD), Consejo Superior de Investigaciones Científicas (CSIC), Carretera Montañana 1005, 50059 Zaragoza, Spain. Electronic address: picorel@eead.csic.es.
authors.forename: Rafael
authors.initials: R
authors.lastname: Picorel
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D046939:Synechocystis / Q000166:cytology / Q000201:enzymology*
chemical_list: D003573:Cytochrome b Group; D045332:Photosystem II Protein Complex; D021122:Protein Subunits; D008320:Maltose; C019313:cytochrome b559
keywords: Alpha-helix; Chimeric protein; Cytochrome; Membrane; Photosynthesis; Protein assembly