Heme-protein vibrational couplings in cytochrome c provide a dynamic link that connects the heme-iron and the protein surface

Abstract

The active site of cytochrome c (Cyt c ) consists of a heme covalently linked to a pentapeptide segment (Cys-X-X-Cys-His), which provides a link between the heme and the protein surface, where the redox partners of Cyt c bind. To elucidate the vibrational properties of heme c , nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57 Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c 552 , including 13 C 8 -heme–, 13 C 5 15 N-Met–, and 13 C 15 N-polypeptide (pp)–labeled samples, revealing heme-based vibrational modes in the 200- to 450-cm −1 spectral region. Simulations of the NRVS spectra of H. thermophilus cytochrome c 552 allowed for a complete assignment of the Fe vibrational spectrum of the protein-bound heme, as well as the quantitative determination of the amount of mixing between local heme vibrations and pp modes from the Cys-X-X-Cys-His motif. These results provide the basis to propose that heme-pp vibrational dynamic couplings play a role in electron transfer (ET) by coupling vibrations of the heme directly to vibrations of the pp at the protein–protein interface. This could allow for the direct transduction of the thermal (vibrational) energy from the protein surface to the heme that is released on protein/protein complex formation, or it could modulate the heme vibrations in the protein/protein complex to minimize reorganization energy. Both mechanisms lower energy barriers for ET. Notably, the conformation of the distal Met side chain is fine-tuned in the protein to localize heme-pp mixed vibrations within the 250- to 400-cm −1 spectral region. These findings point to a particular orientation of the distal Met that maximizes ET.

Alternative ground states enable pathway switching in biological electron transfer

“…After the ET reaction takes place, the reduced state does not exhibit an energy minimum of π u symmetry, so the system relaxes toward the σ u * ground state, which is optimized for subsequent intramolecular ET reactions. In line with this proposal, a recent study has provided evidence of a direct vibrational coupling between an ET metal site and the protein surface, suggesting that formation of a transient interprotein complex could be transmitted to the metal site (27). Moreover, transient binding of redox protein partners is known to modify the reduction potential of donor and acceptor sites (28) and to induce geometrical distortions of copper redox centers (29,30).…”

Investigations of heme distortion, low-frequency vibrational excitations, and electron transfer in cytochrome c

“…S2.3. As discussed previously (23), the lowfrequency spectra reflect coupling between the heme vibrations, the axial ligands, and the CXXCH motif. Thus, the change of the axial Met ligand orientation from R-Met in hh cyt c to S-Met in Pa cyt c 551 along with the slightly different CXXCH configuration are likely responsible for subtle changes of the low-frequency modes.…”

“…In addition to introducing anharmonicity (37), this mixing can tune the mode-mode coupling resonances and allow interactions between the electron transfer partners that might affect the electron transfer rate (23). Moreover, thermal excitations of the ruffling mode, which effectively modulates the donor-acceptor distance, are expected to contribute to the temperature dependence of the rate (41).…”

“…The CXXCH pentapeptide in cyt c may be critical to the ruffled structure and the function of cyt c (2,3,23). The CXXCH unit is thought to affect the heme reduction potential (1), and it can influence heme deformation through the covalent bonding of the thioether groups and by hydrogen bonding within the pentapeptide (2,3).…”

“…Furthermore, the CXXCH pentapeptide may have a biologically important role related to its proximity to the electron transfer partner binding site, as in the yeast cyt c peroxidase/cyt c complex (24). The local vibrational modes of heme in the 250-400 cm −1 region have been shown to strongly mix with the vibrational modes of the CXXCH motif (23). This suggests that the heme-CXXCH vibrational dynamic couplings can play a role in electron transfer by coupling the vibrations of the heme directly to vibrations of the CXXCH unit at the protein-protein interface.…”