Effects of Mutational (Lys to Ala) Surface Charge Changes on the Redox Properties of Electrode-Immobilized Cytochrome c

Abstract

Untrimethylated yeast iso-1-cytochrome c (cytc) and its single and multiple Lys to Ala variants at the surface lysines 72, 73, and 79 were adsorbed on carboxyalkanethiol self-assembled monolayers (SAMs) on gold, and the thermodynamics and kinetics of the heterogeneous protein-electrode electron-transfer (ET) reaction were determined by voltammetry. The reaction thermodynamics were also measured for the same species freely diffusing in solution. The selected lysine residues surround the heme group and contribute to the positively charged domain of cytc involved in the binding to redox partners and to carboxyl-terminated SAM-coated surfaces. The E degrees' (standard reduction potential) values for the proteins immobilized on SAMs made of 11-mercapto-1-undecanoic acid and 11-mercapto-1-undecanol on gold were found to be lower than those for the corresponding diffusing species owing to the stabilization of the ferric state by the negatively charged SAM. For the immobilized proteins, Lys to Ala substitution(s) do not affect the surface coverage, but induce significant changes in the E degrees' values, which do not simply follow the Coulomb law. The results suggest that the species-dependent orientation of the protein (and thereby of the heme group) toward the negatively charged SAM influences the electrostatic interaction and the resulting E degree' change. Moreover, these charge suppressions moderately affect the kinetics of the heterogeneous ET acting on the reorganization energy and the donor-acceptor distance. The kinetic data suggest that none of the studied lysines belong to the interfacial ET pathway.

The effects of ATP and sodium chloride on the cytochrome c–cardiolipin interaction: The contrasting behavior of the horse heart and yeast proteins

“…Furthermore, yeast cyt c has a larger dipole moment than the horse protein (531 Debye in yeast and 253 Debye in horse cyt c, as calculated using the Protein Dipole Moments Server described in [42]). Dipole moment values have been shown to be an important factor in determining the interaction of cyt c on charged surfaces [43], and electrostatic interactions of the former protein with the negatively charged phosphate groups of CL might be very strong and less affected by ionic strength. Clearly, further investigation is necessary to clarify this point.…”

The impact of urea-induced unfolding on the redox process of immobilised cytochrome c

“…Conversely, the entropic term DS 0 rc disfavours protein reduction, yielding a negative contribution to the E°0 values. Here, solvent reorganisation effects, charge redistribution and changes in protein flexibility associated with the haem reduction play the major role in determining DS 0 rc [25,26,35,[56][57][58][59][60][61][62]. At increasing urea concentration DS 0 rc and DH 0 rc of both proteins shift towards negative values (Table 1).…”

“…Protein adsorption on the SAM-coated gold electrode was achieved by dipping the functionalised electrode into a 0.2 mM protein solution at 277 K for 5 h. Standard electrolyte solutions included 5 mM sodium perchlorate and 5 mM phosphate buffer at pH 7. The urea concentration was varied between 0 and 8 M. The formal reduction potentials E°0 were taken to be the midpoint between the anodic and cathodic peak potentials [26] and were found to be almost independent of scan rate in the range 0.02-5 V s -1 . For each species, the experiments were performed at least five times and the reduction potentials were found to be reproducible within ±2 mV.…”

“…The k s values obtained from five measurements were found to be reproducible within 6%. The CV experiments at different temperatures were carried out with a cell in a ''non-isothermal'' setting [26], in which the reference electrode was kept at constant temperature (294 ± 0.1 K) whereas the half-cell containing the working electrode and the Vycor Ò junction to the reference electrode was under thermostatic control with a water bath. The temperature was varied from 278 to 323 K. With this experimental configuration, the standard entropy change for Fe(III) to Fe(II) cytochrome c reduction ðDS 0 rc Þ is given by [27,28] …”

“…The enthalpy term DH 0 rc is considered to be the most important for the high reduction potentials of cytochromes c. It is mainly the result of stabilising the ferrous form owing to ligand binding interactions, the hydrophobicity in the haem pocket, and the limited solvent accessibility [2,3,35]. The electrostatic interactions of the charge of the redox centre with buried and surfaces charges, polar groups of the protein, solvent dipoles and the ionic environment are additional important factors constituting the enthalpic term [2,3,26,35,[52][53][54][55]. Conversely, the entropic term DS 0 rc disfavours protein reduction, yielding a negative contribution to the E°0 values.…”