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

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 ⁵⁷Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c₅₅₂, including ¹³C₈-heme-, ¹³C₈ ¹⁵N-Met-, and ¹³C₈ ¹⁵N-polypeptide (pp)–Habeled samples, revealing heme-based vibrational modes in the 200-to 450-cm⁻¹ spectral region. Simulations of the NRVS spectra of H. thermophilus cytochrome c₅₅₂ 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-XCys-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.