The recombinant di-heme cytochrome c4 from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature an hydroxide ion axially bound to the heme iron at the N- and C-terminal domain, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups facilitates analysis of the heterogeneous and intra-heme electron transfer for these two-centered systems in which the high- and low-potential heme are swept over in the bilobal protein framework. The voltammetric behavior of these species, which experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode, unequivocally shows that intra-heme electron transfer is activated only in the immobilized proteins, as proposed previously for the homologous species from Pseudomonas stutzeri. T-dependent kinetic measurements show that for both proteins the C-lobe faces the HOOC-terminated SAM-coated electrode at a distance of slightly more than 7 Å. The reduction thermodynamics for the native and mutated heme (measured for the first time for a di-heme cytochrome c) in the diffusing regime reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. Protein-electrode orientation and efficient intra-heme ET enable the His,OH--ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes an unprecedented two-centered heme-base biocatalytic interface to be exploited for “third-generation” amperometric biosensing.

Heterogeneous Electron Transfer of a Two-Centered Heme Protein: Redox and Electrocatalytic Properties of Surface-Immobilized Cytochrome c4 / Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; DI ROCCO, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco. - In: THE JOURNAL OF PHYSICAL CHEMISTRY. B. - ISSN 1520-5207. - STAMPA. - 113:41(2009), pp. 13645-13653. [10.1021/jp906339u]

Heterogeneous Electron Transfer of a Two-Centered Heme Protein: Redox and Electrocatalytic Properties of Surface-Immobilized Cytochrome c4

MONARI, Stefano;BATTISTUZZI, Gianantonio;BORSARI, Marco;DI ROCCO, Giulia;MARTINI, LAURA;RANIERI, Antonio;SOLA, Marco
2009

Abstract

The recombinant di-heme cytochrome c4 from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature an hydroxide ion axially bound to the heme iron at the N- and C-terminal domain, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups facilitates analysis of the heterogeneous and intra-heme electron transfer for these two-centered systems in which the high- and low-potential heme are swept over in the bilobal protein framework. The voltammetric behavior of these species, which experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode, unequivocally shows that intra-heme electron transfer is activated only in the immobilized proteins, as proposed previously for the homologous species from Pseudomonas stutzeri. T-dependent kinetic measurements show that for both proteins the C-lobe faces the HOOC-terminated SAM-coated electrode at a distance of slightly more than 7 Å. The reduction thermodynamics for the native and mutated heme (measured for the first time for a di-heme cytochrome c) in the diffusing regime reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. Protein-electrode orientation and efficient intra-heme ET enable the His,OH--ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes an unprecedented two-centered heme-base biocatalytic interface to be exploited for “third-generation” amperometric biosensing.
2009
113
41
13645
13653
Heterogeneous Electron Transfer of a Two-Centered Heme Protein: Redox and Electrocatalytic Properties of Surface-Immobilized Cytochrome c4 / Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; DI ROCCO, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco. - In: THE JOURNAL OF PHYSICAL CHEMISTRY. B. - ISSN 1520-5207. - STAMPA. - 113:41(2009), pp. 13645-13653. [10.1021/jp906339u]
Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; DI ROCCO, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/622627
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