The 16 kDa di-heme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in E. coli and investigated through UV-Vis, MCD and 1H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides di-heme cytochrome c (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 -helices and a compact overall folding. The C-terminal domain is less helical, as the corresponding domain of R. sphaeroides DHC. The two heme groups are bridged by Tyr26 in correspondence of 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 pKa values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and ionization of the heme propionate-7, respectively. Reduction potentials of -0.144 and -0.257 V (vs SHE), were determined for the C- and N-terminal heme group, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and found to reproduce successfully the ionic strength dependence of E°’.

Cloning, Expression and Physico-Chemical Characterization of a New Di-Heme Cytochrome c from Shewanella baltica OS155 / Di Rocco, Giulia; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Borsari, Marco; Ferrari, Erika; Monari, Stefano; Sola, Marco. - In: JBIC. - ISSN 0949-8257. - STAMPA. - 16:(2011), pp. 461-471. [10.1007/s00775-010-0742-y]

Cloning, Expression and Physico-Chemical Characterization of a New Di-Heme Cytochrome c from Shewanella baltica OS155.

DI ROCCO, Giulia;BATTISTUZZI, Gianantonio;BORTOLOTTI, Carlo Augusto;BORSARI, Marco;FERRARI, Erika;MONARI, Stefano;SOLA, Marco
2011

Abstract

The 16 kDa di-heme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in E. coli and investigated through UV-Vis, MCD and 1H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides di-heme cytochrome c (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 -helices and a compact overall folding. The C-terminal domain is less helical, as the corresponding domain of R. sphaeroides DHC. The two heme groups are bridged by Tyr26 in correspondence of 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 pKa values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and ionization of the heme propionate-7, respectively. Reduction potentials of -0.144 and -0.257 V (vs SHE), were determined for the C- and N-terminal heme group, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and found to reproduce successfully the ionic strength dependence of E°’.
16
461
471
Cloning, Expression and Physico-Chemical Characterization of a New Di-Heme Cytochrome c from Shewanella baltica OS155 / Di Rocco, Giulia; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Borsari, Marco; Ferrari, Erika; Monari, Stefano; Sola, Marco. - In: JBIC. - ISSN 0949-8257. - STAMPA. - 16:(2011), pp. 461-471. [10.1007/s00775-010-0742-y]
DI ROCCO, Giulia; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Borsari, Marco; Ferrari, Erika; Monari, Stefano; Sola, Marco
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11380/645610
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