Nanoscopic and Redox Characterization of Engineered Horse Cytochrome c Chemisorbed on a Bare Gold Electrode

Abstract

In this paper, we exploit the potential offered by site-directed mutagenesis to achieve direct adsorption of horse cyt c on a bare gold electrode surface. To this issue, the side chain T102 has been replaced by a cysteine. T102 is close to the surface exposed C-terminal residue (E104), therefore the T102C mutation is expected to generate an exposed cysteine side chain able to facilitate protein binding to the electrode via the sulphur atom (analogously to what observed for yeast iso-1-cyt c). Scanning Tunnelling and Tapping Mode Atomic Force Microscopy measurements show that the T102C mutant stably adsorbs on an Au(111) surface and retains the morphological characteristics of the native form. Cyclic voltammetry reveals that the adsorbed variant is electroactive; however, the heterogeneous electron transfer with the electrode surface is slower than that observed for yeast iso-1-cyt c. We ascribe it to differences in the tertiary architecture of the two proteins, characterized by different flexibility and stability. In particular, the region where the N- and C-terminal helices get in contact (and where the mutation occurs) is analyzed in detail, since the interactions between these two helices are considered crucial for the stability of the overall protein fold.

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…Site-directed mutagenesis has been used to introduce cysteine residues on the surface of blue copper proteins (azurin and plastocyanin), thus facilitating protein chemisorption on unmodified gold. – When this strategy was used to promote direct ET to a surface cysteine mutant of Alcaligenes faecalis copper-containing nitrite reductase , only poor catalytic activity could be detected (although some noncatalytic signals were observed); pyrolytic graphite edge eventually proved to be a better choice for studying this enzyme. , In contrast, cytochrome c nitrite reductase (ccNiR) retains activity when it is immobilized onto an unmodified Au(111) surface . Heering and coworkers used the reactivity of two thiols present at the surface of a ferredoxin from Pyroccocus furiosus to directly immobilize the protein on a bare gold electrode, with an orientation such that the cluster is exposed to solution, providing an electrode surface with docking sites for the potential attachment of redox enzymes (see section 3.1.1.7 below).…”

“…Site-directed mutagenesis has been used to introduce cysteine residues on the surface of blue copper proteins (azurin and plastocyanin), thus facilitating protein chemisorption on unmodified gold. [212][213][214][215] When this strategy was used to promote direct ET to a surface cysteine mutant of Alcaligenes faecalis copper-containing nitrite reductase, only poor catalytic activity could be detected (although some noncatalytic signals were observed); 216 pyrolytic graphite edge eventually proved to be a better choice for studying this enzyme. 167,185 In contrast, cytochrome c nitrite reductase (ccNiR) retains activity when it is immobilized onto an unmodified Au(111) surface.…”

Biosensor based on chemically-designed anchorable cytochrome c for the detection of H2O2 released by aquaticcells

“…In that context, the possibility to modify the cyt c protein shell with promoters which facilitate its chemisorption and enhance ET rate constant has been scarcely reported. By analogy to the yeast iso-1-cyt c (cyt c) which contains a cystein in position C102 (Bortolotti et al, 2006;Heering et al, 2004), Andolfi et al (2007) succeeded in replacing the side chain T102 of cyt c with a cystein via site-directed mutagenesis but the resulting mutant remained electroinactive.…”

Engineered Proteins: Redox Properties and Their Applications

“…Furthermore, direct electrical communication between the redox centers and the metal electrodes yielded very good electrochemical performances without dramatically affecting the structural properties of the bound proteins, especially for the Cyt c variants whose redox potential could be reversibly modulated (23). This important achievement represented a series of possible suitable and appealing candidates for constituting the biocatalytic interface in biomolecular electronic applications, with more potential in nanostructured biosensing devices (3).…”