Effects of Protein Structure on Iron–Polypeptide Vibrational Dynamic Coupling in Cytochrome c

• Published in: Biochemistry (Easton) Vol. 54; no. 4; pp. 1064 - 1076
• Main Authors: Galinato, Mary Grace I, Bowman, Sarah E. J, Kleingardner, Jesse G, Martin, Sherri, Zhao, Jiyong, Sturhahn, Wolfgang, Alp, E. Ercan, Bren, Kara L, Lehnert, Nicolai
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.02.2015; American Chemical Society (ACS)
• Subjects: BASIC BIOLOGICAL SCIENCES; Binding Sites - physiology; Cytochromes c - chemistry; Cytochromes c - metabolism; Hemoglobins - chemistry; Hemoglobins - metabolism; Peptides - chemistry; Peptides - metabolism; Protein Structure, Secondary; Vibration
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi501430z

Cytochrome c (Cyt c) has a heme covalently bound to the polypeptide via a Cys-X-X-Cys-His (CXXCH) linker that is located in the interface region for protein–protein interactions. To determine whether the polypeptide matrix influences iron vibrational dynamics, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c-552, and variants M13V, M13V/K22M, and A7F, which have structural modifications that alter the composition or environment of the CXXCH pentapeptide loop. Simulations of the NRVS data indicate that the 150–325 cm–1 region is dominated by NHis–Fe–SMet axial ligand and polypeptide motions, while the 325–400 cm–1 region shows dominant contributions from ν­(Fe–NPyr) (Pyr = pyrrole) and other heme-based modes. Diagnostic spectral signatures that directly relate to structural features of the heme active site are identified using a quantum chemistry-centered normal coordinate analysis (QCC-NCA). In particular, spectral features that directly correlate with CXXCH loop stiffness, the strength of the Fe–His interaction, and the degree of heme distortion are identified. Cumulative results from our investigation suggest that compared to the wild type (wt), variants M13V and M13V/K22M have a more rigid CXXCH pentapeptide segment, a stronger Fe–NHis interaction, and a more ruffled heme. Conversely, the A7F variant has a more planar heme and a weaker Fe–NHis bond. These results are correlated to the observed changes in reduction potential between wt protein and the variants studied here. Implications of these results for Cyt c biogenesis and electron transfer are also discussed.