PH-LINKED CONFORMATIONAL REGULATION OF A METALLOPROTEIN OXIDATION REDUCTION EQUILIBRIUM - ELECTROCHEMICAL ANALYSIS OF THE ALKALINE FORM OF CYTOCHROME-C

The direct electrochemistry of a variety of cytochromes c at pyrolytic graphite surfaces has been studied by cyclic voltammetry over a wide range of sweep rate and over a range of solution pH in which ferricytochrome c can exist in either the native or alkaline conformations. The electrochemistry observed is entirely consistent with both conformers of cytochrome c being fully electroactive at this electrode surface. Experiments at sweep rates of 1 V s-1 and above show that both cytochrome conformers behave as simple one-electron-transfer proteins. This finding has provided the first measurement of the midpoint reduction potential for the alkaline form of cytochrome c and thereby allows a complete thermodynamic analysis of the alkaline conformational cycle involving both oxidation states of the two conformers of the protein. Central to the evidence provided for this analysis are the results obtained from site-specific variants of yeast iso-1-cytochrome c which have altered alkaline pK(a)s. The reduction potential of the alkaline form of wild-type yeast iso-1-cytochrome c at pH 8.45 is -205 mV vs SHE. The same value is obtained for the equivalent conformer of the horse heart protein at pH 10.0. Experiments at lower sweep rates are consistent with the rearrangement of the reduced alkaline conformer to the reduced native conformer, and the rates of this interconversion have been estimated from the sweep rate dependence for all the cytochromes studied here. This study highlights the role for dynamic electrochemical analysis in the study of cytochrome c and its variants which undergo significant oxidation state linked conformational changes.