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.