Anion Binding to Mitochondrial Cytochromes c Studied through Electrochemistry

Abstract

The redox potential of horse and bovine heart cytochromes c determined through cyclic voltammetry is exploited to probe for anion-protein interactions, using a Debye-Hiickel-based model. In parallel, protein charge neutralization resulting from specific anion binding allows monitoring for surface-charge/ E" relationships. This approach shows that a number of anions, most of which are of biological relevance, namely C1-, HPO:-, HCO;, NO;, SO:-, ClO;, citrate'-and oxalate2-,. bind specifically to the protein surface, often in a sequential manner as a result of the presence of multiple sites with different affinities. The binding stoichiometries of the various anions toward a given cytochrome are in general different. Chloride and phosphate appear to bind to a greater extent to both proteins as compared to the other anions. Differences in binding specificity toward the two cytochromes, although highly sequence-related, are observed for a few anions. The data are discussed comparatively in terms of electrostatic and geometric properties of the anions and by reference to the proposed location and amino acid composition of the anion binding sites, when available. Specific binding of this large set of anions bearing different charges allows the electrostatic effect on E" due to neutralization of net positive protein surface charge(s) to be monitored. 'H NMR indeed indicates the absence of significant salt-induced structural perturbations, hence the above change in ED is predominantly electrostatic in origin. A systematic study of protein surface-chargelE" relationships using this approach is unprecedented. Values of 15 -25 mV (extrapolated at zero ionic strength) are obtained for the decrease in E" due to neutralization of one positive surface charge, which are of the same order of magnitude as previous estimates obtained with either mutation or chemical modification of surface lysines. The effects of the anion-induced decrease of net positive charge on ED persist also at a relatively high ionic strength and add to the general effects related to the charge shielding of the protein as a whole due to the surrounding ionic atmosphere: hence the ionic strength dependence of the rate of electron transfer between cytochromes c and redox partners could also involve salt-induced changes in the driving force.Keywords: cytochrome c ; electrochemistry; redox potential ; protein charge; protein dielectric.Electron flow in biological systems is still, after decades, one of the leading areas of biophysical research [I -41. Secondorder collision-dependent processes received much attention, particularly regarding the thermodynamic and mechanistic aspects of the formation of the precursor complex and the determi- relevance to these proteins in terms of affinity constants, location of the sites of specific binding and number of anions bound to the protein [15, 16,[23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. The results are somewhat heterogeneous, due to the different techniques and conditions employed and to the int...

Redox thermodynamics of cytochrome c adsorbed on mercaptoundecanol monolayer electrodes

“…The temperature dependences of E 00 allowed to obtain the corresponding thermodynamic parameters of the redox process ( Table 1). The data are in line with previous reports [7,48,49] and confirm the different abilities of the anions to interact with the surface of the cytochrome in solution [50].…”

“…This effect probably arises from the partial neutralization of positive charges of the protein by carboxylate group(s) existing at the surface of the SAM arising from mercaptoundecanoic acid. In fact, for a protein with a net positive charge (as cytochrome c) a stabilization of the oxidized state leading to a decrease of the E 00 value occurs with de- creasing positive surface charges [48,[50][51][52][53]. In the case of the alkanethiolate SAM, an anomalous peak broadening was observed in CV curves of adsorbed cytochrome c, which appeared indicative of a dispersion in E 00 values due to a certain heterogeneity of the adsorbed sites [21] of the monolayer.…”

“…The CV curves of bovine cytochrome c in solution in the presence of different base electrolytes show a single electrochemically reversible, one electron and diffusion controlled voltammetric wave [7,48,49]. The temperature dependences of E 00 allowed to obtain the corresponding thermodynamic parameters of the redox process ( Table 1).…”

“…As a consequence, this state will be preferentially stabilized over the other. This occurs for the oxidized state in cytochromes c leading to a decrease of the redox potential with increasing ionic strength [48,[50][51][52][53]. Using a combination of the extended Debye-H€ uckel and Nernst equations, a quantitative model describing E 00 as a function of the ionic strength (I) can be obtained [3].…”

“…The redox potential of cytochrome c in solution is affected by the ionic strength of the medium as a consequence of two main salt-induced effects, namely changes in the activity coefficients of the oxidized and reduced forms and specific anion binding to protein surface sites (at neutral pH cytochromes c bears a net positive charge) [3,48,[50][51][52][53]. The former effect is related to the shielding of the protein charge by the surrounding ''ionic atmosphere'' which is greater for the redox state carrying the larger charge.…”