title: Cloning, expression, and physicochemical characterization of a new diheme cytochrome c from Shewanella baltica OS155.
abstract: The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV-vis, magnetic circular dichroism, and (1)H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid-base equilibria with pK (a) values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of -0.144 and -0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°'.
authors.affiliation: Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41125, Modena, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Di Rocco
authors.forename: Gianantonio
authors.initials: G
authors.lastname: Battistuzzi
authors.forename: Carlo Augusto
authors.initials: CA
authors.lastname: Bortolotti
authors.forename: Marco
authors.initials: M
authors.lastname: Borsari
authors.forename: Erika
authors.initials: E
authors.lastname: Ferrari
authors.forename: Stefano
authors.initials: S
authors.lastname: Monari
authors.forename: Marco
authors.initials: M
authors.lastname: Sola
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004563:Electrochemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D010088:Oxidoreductases; C090099:diheme cytochrome c