title: Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c7 family.
abstract: Cytochromes c(7) are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins-phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c(7) family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of (1)H-(15)N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.
authors.affiliation: Requimte-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus Caparica, 2829-516 Caparica, Portugal.
authors.forename: Joana M
authors.initials: JM
authors.lastname: Dantas
authors.forename: Leonor
authors.initials: L
authors.lastname: Morgado
authors.forename: Yuri Y
authors.initials: YY
authors.lastname: Londer
authors.forename: Ana P
authors.initials: AP
authors.lastname: Fernandes
authors.forename: Ricardo O
authors.initials: RO
authors.lastname: Louro
authors.forename: P Raj
authors.initials: PR
authors.lastname: Pokkuluri
authors.forename: Marianne
authors.initials: M
authors.lastname: Schiffer
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Salgueiro
mesh_terms: D000595:Amino Acid Sequence; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045287:Desulfuromonas / Q000737:chemistry; D045302:Geobacter / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000378:metabolism*; D016415:Sequence Alignment; D013816:Thermodynamics
chemical_list: D003574:Cytochrome c Group; D010649:Phenylalanine