Charge transfer (CT) transitions between the C-terminal carboxylate and peptide group have been investigated for alanyl-X and X-alanine dipeptides by far-UV absorption and electronic circular dichroism (ECD) spectroscopy (where X represents different amino acid residues). The spectra used in the present study were obtained by subtracting the spectrum of the cationic species from that of the corresponding zwitterionic peptide spectrum. These spectra displayed three bands, e.g., band I between 44 and 50 kK (kK = 10(3) cm(-1)), band II at 53 kK, and band III above 55 kK, which were, respectively, assigned to a n(COO-) --> pi* CT transition, a pi(COO-) --> pi* CT transition, and a carboxylate pi --> pi* (NV1) transition, respectively By comparison of the intensity, bandwidth, and wavenumber position of band I of some of the investigated dipeptides, we found that positive charges on the N-terminal side chain (for X = K), and to a minor extent also the N-terminal proton, reduce its intensity. This can be understood in terms of attractive Coulomb interactions that stabilize the ground state over the charge transfer state. For alanylphenylalanine, we assigned band I to a n(COO-) --> pi* CT transition into the aromatic side chain, indicating that aromatic side chains interact electronically with the backbone. We also performed ECD measurements at different pH values (pH 1-6) for a selected subset of XA and AX peptides. By subtraction of the pH 1 spectrum from that observed at pH 6, the ECD spectrum of the CT transition was obtained. A titration curve of their spectra reveals a substantial dependence on the protonation state of the aspartic acid side chain of AD, which is absent in DA and AE. This most likely reflects a conformational transition of the C-terminus into a less extended state, though the involvement of a side chain --> peptide CT transition cannot be completely ruled out.

The charge transfer (CT) band at 695 nm in the spectrum of ferri-cytochrome c is highly asymmetric, indicating conformational heterogeneity due to the coexistence of different conformational substates. We have measured the respective band profile of horse heart ferri-cytochrome c as a function of temperature between 283 K (10 degrees C) and 333 K (60 degrees C) and found that the well-known decrease of the absorptivity is wavenumber-dependent and exhibits a biphasic behavior. This indicates that the underlying conformational substates differ in their thermodynamic stability with respect to the structural changes associated with the disappearance of the 695 nm band, which eventually (at high temperatures) involves the replacement of M80 by a nearby lysine residue. Our data further indicates that the thermal unfolding process involves two structurally different intermediate states.