Screen-printed graphite macroelectrodes for the direct electron transfer of cytochrome c: a deeper study of the effect of pH on the conformational states, immobilization and peroxidase activity

Abstract

The direct electron transfer of cytochrome c has been studied at screen-printed graphite macroelectrodes without recourse to mediators or the need for any electrode pre-treatment as is commonly employed within the literature. A wide range of pH values from 2.0 to 11.0 have been explored upon the electrochemical response of cytochrome c and different voltammetric signatures have been observed. The direct electron transfer of the alkaline transition of cytochrome c was found impeded within alkaline media leading to either an irreversible redox process or even no voltammetric responses. In acidic aqueous media the electrochemical process is observed to undergo a mixed diffusion and adsorption controlled process rather than a purely diffusional process of the native conformation as observed at pH 7.0. Interestingly, at pH 3.5 a new conformational state is revealed in cooperation with the native conformation. The immobilization of the protein was satisfactorily obtained using a simple method by cycling the protein at specific solution pH values allowing amperometric responses to be obtained and gives rise to useful pseudo-peroxidase activity for sensing H2O2. Apparent Michaelis-Menten constant values (Km) were calculated via the Lineweaver-Burk method with deduced values of 25 ± 4, 98 ± 12 and 230 ± 30 mM, respectively for pH values of 2.0, 3.0 and 7.0. Such work is important for those utilising cytochrome c in bio-electrochemical and related applications.

Effect of confinement of horse heart cytochrome c and formate dehydrogenase from Candida boidinii on mesoporous carbons on their catalytic activity

“…Cytochromes are redox proteins that transfer electrons by oxidation and reduction processes via the heme group (iron bound to a porphyrin) ( vide supra ). Thus, during the negative scan in the cyclic voltammograms recorded on the prepared Cyt-c/Gx/GCE electrodes, Fe(III) can be reduced to Fe(II) and then oxidized back to Fe (III) during the reverse positive scan [ 40 ].…”

“…To evaluate if the confinement in the mesopores affects the conformation state of Cyt c, the electrochemical response of Cyt-c/Gx/GCE electrodes was investigated in 0.1 M PB at pH 4.0. Indeed, pH is known to have a strong effect in the conformational changes in the heme group of Cyt c, with consequences on the electron transfer kinetic and therefore on the pseudo-peroxidise activity of the protein [ 40 , 44 – 46 ]. Cyt c may exist in solution in five reversible pH-dependent conformational states: I, II, III, IV, and V with pKa values of 0.42, 2.50, 9.35, and 12.76, respectively [ 40 , 47 ].…”

Electrochemical Deposition of Protein‐conjugated Graphene by Pulse Reverse Technique

“…This potential shift can be rationalized in terms of the effect of the negatively charged COO − groups of GO being in close proximity to the heme group of positively charged Cyt c (pI = 10.5) and is in good agreement with the previously reported 174 mV vs . Ag/AgCl for Cyt c on screen‐printed graphite electrodes . The surface coverage ( Γ ) of Cyt c can be estimated according to Laviron's equation: where, Γ (mol/cm 2 ) is the average amount of adsorbed Cyt c on the electrode surface, A is the electrode area (cm 2 ), n is the number of electrons transferred, and F , R , and T each have their usual significance.…”

“…25 This potential shift can be rationalized in terms of the effect of the negatively charged COO − groups of GO being in close proximity to the heme group of positively charged Cyt c (pI = 10.5) and is in good agreement with the previously reported 174 mV vs. Ag/AgCl for Cyt c on screen-printed graphite electrodes. 26 The surface coverage (Γ) of Cyt c can be estimated according to Laviron's equation 27 :…”

Effects of Film Morphology and Surface Chemistry on the Direct Electrochemistry of Cytochrome c at Boron-Doped Diamond Electrodes

“…These are the first such ΔE p -pH data (6–9) reported for this redox protein at diamond electrodes. In recent work with screen-printed graphite electrodes, researchers have shown that direct electron transfer in aqueous solutions is possible due to the electrostatic attraction between the positively-charged lysine goups on the heme protein and the negatively-charged carboxylate functional groups on the electrode surface [72,73]. The authors attributed the observed pH dependence of the electron-transfer kinetics to changes in the protein orientation and distance of closest approach at the electrode surface.…”