ANION-BINDING TO MITOCHONDRIAL CYTOCHROME-C STUDIED THROUGH ELECTROCHEMISTRY - EFFECTS OF THE NEUTRALIZATION OF SURFACE-CHARGES ON THE REDOX POTENTIAL

The redox potential of horse and bovine heart cytochromes c determined through cyclic voltammetry is exploited to probe for anion-protein in teractions, using a Debye-Huckel-based model. In parallel, protein cha rge neutralization resulting from specific anion binding allows monito ring for surface-charge/E(o) relationships. This approach shows that a number of anions, most of which are of biological relevance, namely C l-, HPO42-, HCO3-, NO3-, SO42-, ClO4-, citrate(3-) and oxalate(2-); bi nd specifically to the protein surface, often in a sequential manner a s a result of the presence of multiple sites with different affinities , The binding stoichiometries of the various anions toward a given cyt ochrome are in general different. Chloride and phosphate appear to bin d to a greater extent to both proteins as compared to the other anions . Differences in binding specificity toward the two cytochromes, altho ugh highly sequence-related, are observed for a few anions. The data a re discussed comparatively in terms of electrostatic and geometric pro perties of the anions and by reference to the proposed location and am ino acid composition of the anion binding sites, when available. Speci fic binding of this large set of anions bearing different charges allo ws the electrostatic effect on E(o) due to neutralization of net posit ive protein surface charge(s) to be monitored. H-1 NMR indeed indicate s the absence of significant salt-induced structural perturbations, he nce the above change in E(o) is predominantly electrostatic in origin. A systematic study of protein surface-charge/E(o) relationships using this approach is unprecedented. Values of 15-25 mV (extrapolated at z ero ionic strength) are obtained for the decrease in E(o) due to neutr alization of one positive surface charge, which are of the same order of magnitude as previous estimates obtained with either mutation or ch emical modification of surface lysines. The effects of the anion-induc ed decrease of net positive charge on E(o) persist also at a relativel y high ionic strength and add to the general effects related to the ch arge shielding of the protein as a whole due to the surrounding ionic atmosphere: hence the ionic strength dependence of the rate of electro n transfer between cytochromes c and redox partners could also involve salt-induced changes in the driving force.