Involvement of lysines-72 and -79 in the alkaline isomerization of horse heart ferricytochrome c

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Abstract
Spectrophotometric titrations of 5 singly modified horse heart ferricytochromes c, specifically (trifluoromethyl)phenylcarbamylated (CF3PhNHCO.sbd.) or trifluoroacetylated (CF3CO.sbd.) at lysines-13, -72 and -79 were carried out. The CF3PhNHCO-Lys-13, Lys-79 and CF3CO-Lys-79 derivatives all underwent alkaline isomerization with loss of the 695-nm band to low-spin species with an apparent pK of about 8.9, as did the unmodified cytochrome. Modification of lysine-72 appeared to alter the reaction pathway since the CF3PhNHCO-Lys-72 derivative isomerized to a high-spin form with an apparent pK of 9.3 while the CF3CO-Lys-72 derivative isomerized to a low-spin species with an apparent pK of 9.6, indicating that lysine-72 may be the normal 6th Fe ligand in the native protein alkaline isomer. A model was suggested for the alkaline transition in which replacement of the methionine Fe ligand is dependent on a number of factors, including the local availability and relative affinities of possible ligands for the heme Fe and the effects of ionic and hydrophobic interactions on the tertiary structure of the molecule.