Surface of Cytochrome<i>c</i>: Infrared Spectroscopy of Carboxyl Groups

Abstract

The carboxylate groups of organic acids give strong absorption in the infrared between approximately 1550 and 1650 cm-1. For acetate and chloroacetate derivatives, the infrared (IR) frequency of the carboxylate antisymmetric stretching mode (v(a)OCO) is related to the square root of the pK of the acid, with a shift of approximately 20 cm-1 to higher frequency for a pK drop in the range 5-3. It follows that v(a)OCO may respond to conditions on the protein surface. In this paper, the IR amide I' and carboxylate absorptions of cytochrome c from horse, yeast, and tuna are compared with model compounds such as Val-Glu and microperoxidase-11, the 11 amino acid fragment of horse cytochrome c containing the covalently bound heme. For microperoxidase-11, the contribution from all four carboxylates can be accounted for and the 1567 cm-1 absorption is assigned to the heme propionates. For the proteins, the carboxylate absorption band is inhomogeneous, i.e., there is a distribution of frequencies. Both the amide I' and carboxylate bands are sensitive to protein conformation as shown by their different pH, salt, and redox dependence.