Vibrational Dynamics of Iron in Cytochrome c

Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts 02115, USA.
The Journal of Physical Chemistry B (Impact Factor: 3.38). 02/2009; 113(7):2193-200. DOI: 10.1021/jp806574t
Source: PubMed

ABSTRACT Nuclear resonance vibrational spectroscopy (NRVS) and Raman spectroscopy on (54)Fe- and (57)Fe-enriched cytochrome c (cyt c) identify multiple bands involving vibrations of the heme Fe. Comparison with predictions from Fe isotope shifts reveals that 70% of the NRVS signal in the 300-450 cm(-1) frequency range corresponds to vibrations resolved in Soret-enhanced Raman spectra. This frequency range dominates the "stiffness", an effective force constant determined by the Fe vibrational density of states (VDOS), which measures the strength of nearest-neighbor interactions with Fe. The stiffness of the low-spin Fe environment in both oxidation states of cyt c significantly exceeds that for the high-spin Fe in deoxymyoglobin, where the 200-300 cm(-1) frequency range dominates the VDOS. This situation is reflected in the shorter Fe-ligand bond lengths in the former with respect to the latter. The longer Fe-S(Met80) in oxidized cyt c with respect to reduced cyt c leads to a decrease in the stiffness of the iron environment upon oxidation. Comparison with NRVS measurements allows us to assess assignments for vibrational modes resolved in this region of the heme Raman spectrum. We consider the possibility that the 372 cm(-1) band in reduced cyt c involves the Fe-S(Met80) bond.


Available from: Wolfgang Sturhahn, May 28, 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-vis spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand previously shown to be from HCCS residue His154. Substitutions of His19 in cytochrome c to seven other residues (G,A,M,R,K,C,Y) were used with various approaches to establish other roles played by His19. Three roles for His19 in HCCS-mediated assembly are suggested: i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His19.
    Journal of Biological Chemistry 08/2014; 289(42). DOI:10.1074/jbc.M114.593509 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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. In order to determine whether the polypeptide matrix influences iron vibrational dynamics, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on (57)Fe-labeled 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) 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 are identified that directly correlate with CXXCH loop stiffness, the strength of the Fe-His interaction, and the degree of heme distortion. Cumulative results from our investigation suggest that compared to 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 variant A7F has a more planar heme and a weaker Fe-NHis bond. These results are correlated to the observed changes in redox potential between wt protein and the variants studied here. Implications of these results for Cyt c biogenesis and electron transfer are also discussed.
    Biochemistry 12/2014; 54(4). DOI:10.1021/bi501430z · 3.19 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We use nuclear resonance inelastic x-ray scattering (NRIXS), a relatively new, synchrotron-based, isotope-specific technique in combination with a more traditional one, Raman spectroscopy, to probe the vibrational dynamics of the host frameworks in two Zintl clathrates: K 8Zn4Sn42 (KZS) and Ba8Ga16Sn30 (BGS). From the normalized Sn vibrational density of states obtained from NRIXS, we calculate the stiffness, a mean force constant of the Sn environment, the resilience, a compact way of expressing the temperature dependence of the Sn mean square displacement, and several thermodynamic properties. The stiffness and the resilience are approximately 7% lower in KZS, reflecting its larger unit cell compared to BGS. We emphasize the complementariness between NRIXS and Raman spectroscopy and establish a series of benchmarks for a more quantitative evaluation of the Raman spectra for the numerous clathrates that are still not suitable for NRIXS studies.
    Physical Review B 09/2014; Phys. Rev. B(90):104304. DOI:10.1103/PhysRevB.90.104304 · 3.66 Impact Factor