Identification of a New Charge-Transfer Transition through the Partial Unfolding of Cytochrome C under Mild Acidic Conditions

Abstract

Cytochrome c, a model protein, has been isolated from a wide variety of prokaryotic and eukaryotic sources. It is commonly believed that proteins behave identically if it is extracted from the same source. This investigation illustrates the unexpected conformational change on the exposure of oxidized equine heart cytochrome c to TCA, as opposed to the preparation from acetic acid, both obtained from Sigma-Aldrich and dissolved at a concentration of 0.5 mM in 1.0 mM monobasic phosphate buffer. Without further purification an earlier unfolding event was obtained with the TCA exposed protein (pH 7.0, Temp. 338K). To unravel this enigma a variety of purification methods were employed utilizing partial unfolding-refolding protocols. The complete oxidation of cytochrome c was achieved by adjusting the pH of the sample to 11.5, neutralizing the positive patches on the protein surface. After complete oxidization, the protein was passed over a Sephadex G10 column. Subsequently, the pH of the solution was adjusted back to 8.0, which was the starting point of a titration which covered the region between 8.0 and 4.0. Surprisingly we observed a weak absorption band at 625nm, which increased with decreasing pH, reflecting the protonation of a titratable group (pK=5.2). This value strongly indicates a H33 as the involved residue. Normally, this band is indicative of the population of a ferric high spin state of the heme iron. However, in our case the 695nm band, which is an indicator of the intactness of the Fe(III)-M80 bond, remained present without any loss of intensity. Our observation suggest the stabilization of an excited charge transfer state, possibly resulting from an A2u->dπ transition by the positive charge on H33 in a heme cavity modified by the initially present TCA.