Vibrational dynamics of iron in cytochrome C.
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.
Subscriber access provided by ARGONNE NATL LAB
The Journal of Physical Chemistry B is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Vibrational Dynamics of Iron in Cytochrome c
Bogdan M. Leu, Tom H. Ching, Jiyong Zhao, Wolfgang Sturhahn, E. Ercan Alp, and J. Timothy Sage
J. Phys. Chem. B, 2009, 113 (7), 2193-2200• Publication Date (Web): 27 January 2009
Downloaded from http://pubs.acs.org on March 31, 2009
More About This Article
Additional resources and features associated with this article are available within the HTML version:
Access to high resolution figures
Links to articles and content related to this article
Copyright permission to reproduce figures and/or text from this article
Vibrational Dynamics of Iron in Cytochrome c
Bogdan M. Leu,†,‡Tom H. Ching,†Jiyong Zhao,‡Wolfgang Sturhahn,‡E. Ercan Alp,‡and
J. Timothy Sage*,†
Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern
UniVersity, Boston, Massachusetts 02115, and AdVanced Photon Source, Argonne National Laboratory,
Argonne, Illinois 60439
ReceiVed: July 24, 2008; ReVised Manuscript ReceiVed: December 11, 2008
Nuclear resonance vibrational spectroscopy (NRVS) and Raman spectroscopy on54Fe- and57Fe-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-1frequency 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-1band in reduced cyt c involves the Fe-S(Met80)
Cytochrome c (cyt c) is a relatively small heme protein (104
amino acids, MW ) 12 400 Da) that mediates electron transfer
between two membrane-bound protein complexes in the mito-
chondrial electron-transport chain1-4and also plays a crucial
signaling role in cellular apoptosis.5,6During electron transfer,
the heme iron cycles between Fe(II) and Fe(III). In both
oxidation states, the protein ligates the heme iron by a
methionine (Met80) and a histidine (His18). The heme prosthetic
group connects to the protein via two additional bonds: the
thioether linkages between vinyl groups of the porphyrin ring
and two cysteines (Cys14 and Cys17; Figure 1).
Both oxidation states adopt the same fold at neutral pH, and
the structures are very similar, both in solutions7-11and in
crystals.12-16Structural changes include a small increase of the
Fe-S(Met80) bond length upon oxidation.17Solution structures
derived from NMR measurements reveal a 140 pm rms deviation
between backbone atoms in Fe(II) and Fe(III) cyt c from horse.7,8
Small-angle X-ray scattering studies were interpreted as evi-
dence for an increased radius of gyration for the oxidized state
in low ionic strength solution.18However, more recent analysis
of X-ray scattering from cyt c solutions attributed oxidation state
differences in the small-angle signal from low ionic strength
solutions to unscreened interparticle interactions.19Consistent
with this result, a crystal structure of Fe(III) cyt c at low ionic
strength16resembles that determined at high salt concentration.
Hydrodynamic, thermodynamic, and spectroscopic properties,
as well as ion binding and chemical reactivity, are influenced
by the oxidation state (ref 20 and references therein).
Fe-ligand vibrations are useful probes of the metal environ-
ment in heme proteins but can be difficult to identify in
congested biomolecular vibrational spectra, even with the
selectivity provided by Raman scattering in resonance with the
heme. For cyt c, resonance Raman spectroscopy is a useful tool
for identifying conformational changes,21-24but structural
interpretations of the individual bands remains challenging.
Nuclear resonance vibrational spectroscopy (NRVS) is emerging
as an even more selective vibrational probe, revealing the
complete vibrational density of states (VDOS) for57Fe25-30or
other Mo ¨ssbauer nuclei.31,32
NRVS investigations of myoglobin (Mb) have revealed
Fe-ligand vibrations that contribute weakly to the Raman
signal,33-35presumably because of the restrictive selection rules
that apply to the highly symmetric heme.
In this study, complementary data provided by NRVS and
resonance Raman spectroscopy on reduced
enriched cyt c identify bands in the rich Raman spectra that are
due to heme Fe vibrations. We use Raman frequency shifts upon
isotope substitution to predict the contributions of these modes
to the Fe VDOS. Unexpectedly, comparison with the measured
VDOS reveals that ∼70% of the NRVS signal above 300 cm-1
corresponds to vibrations resolved in the Soret-enhanced Raman
spectrum. On the other hand, NRVS determines the contribu-
tions of these modes to the stiffness, an effective force constant
expected to correlate with Fe-nearest-neighbor bond lengths.
The dominant contributions to the deoxyMb VDOS appear at
lower frequencies (200-300 cm-1), which leads to a lower
stiffness of the heme iron environment in the high-spin deoxyMb
with respect to low-spin cyt c. As expected, stiffness is inversely
related to Fe-ligand bond lengths, the longer Fe-N bonds in
deoxyMb with respect to cyt c being reflected in the lower
stiffness of the former with respect to the latter. On the other
hand, the longer Fe-S bond in Fe(III) cyt c with respect to
* Corresponding author: e-mail email@example.com, phone (617)-373-2908;
‡Argonne National Laboratory.
J. Phys. Chem. B 2009, 113, 2193–2200
10.1021/jp806574t CCC: $40.75
2009 American Chemical Society
Published on Web 01/27/2009
Fe(II) cyt c is the most important contributor to the decreased
stiffness upon oxidation. The maximum NRVS signal of reduced
cyt c corresponds to the biggest shift in the Raman data (372
cm-1) upon isotope substitution and may involve the Fe-S
bond. Stretching of the Fe-N bond to the histidine may
contribute to several vibrations, a situation we encountered
previously in six-coordinated proteins and model compounds.36
In a separate publication,37we used the57Fe-reconstituted protein
to evaluate the mechanical effect of the thioether links. In the
present paper, we explore the vibrational structure in greater
detail, using NRVS measurements on the57Fe-enriched cyt c
together with frequency shifts in the Raman spectrum upon Fe
2. Experimental Methods
2.1. Cyt c Reconstitution with57Fe and54Fe. Because the
heme c in cytochrome c is covalently bonded to the protein via
thioether linkages to Cys14 and Cys17, it cannot easily be
removed as in myoglobin and hemoglobin. We employed the
ferrous sulfate-hydrochloric acid method38to extract the iron
from the heme. We adapted metal reinsertion procedures from
the literature39-41to insert isotopes57Fe and54Fe in the resulting
porphyrin cyt c. The complete preparation procedure is described
2.2.1. NRVS Measurements.57Fe NRVS measurements were
performed at sector 3-ID-D of the Advanced Photon Source at
Argonne National Laboratory as described in detail elsewhere.26
Frozen solutions were loaded into polyethylene sample cups
and mounted on a cryostat cooled by a flow of liquid He, with
X-ray access through a beryllium dome. The incident mono-
chromatic 14.4 keV X-ray had a flux of ∼109Hz, and the
experimental resolution was 8 cm-1(1 meV). Results presented
here are averages of 14-26 energy scans, and comparison of
individual scans found no spectroscopic changes due to radiation
damage. Data analysis described below confirmed temperatures
of 87 K for Fe(II) cyt c and 68 K for Fe(III) cyt c.
2.2.2. Raman Measurements. Raman measurements on
native and57Fe- and54Fe-reconstituted cyt c were performed at
room temperature. Previous Raman measurements on cyt c
found significant line narrowing with reduced temperature, but
reported no frequency changes,43supporting our comparison of
room temperature Raman measurements with cryogenic NRVS
measurements. Raman scattering was excited by the 413.1 nm
line of a krypton laser and detected using a J-Y LabRam HR
Raman microscope. The beam power at the sample was 15 mW.
The frequency calibration was verified using the rich Raman
spectrum of fenchone as a standard. The instrument resolution
was 2.8 cm-1.
2.2.3. Data Analysis. The measured NRVS signal consists
of a central resonance due to recoilless excitation of the57Fe
nuclear excited state at E0) 14.4 keV, accompanied by a series
of sidebands corresponding to creation or annihilation of
vibrational quanta of frequency ν j, displaced from the recoilless
absorption by an energy hcν j.27,44The program PHOENIX,45
working under the assumptions that samples are isotropic,
harmonic, and Debye-like at low energies, removes temperature
Figure 1. Left: cytochrome c (Protein Data Bank89code 1HRC).90This structure was generated with the program RasMol.91Right: cytochrome
c heme. Hydrogen atoms were omitted for clarity. This structure was generated with the program Molekel.92Color scheme: orange ) iron, green
) nitrogen, red ) oxygen, blue ) sulfur, gray ) carbon.
Figure 2. Upper and middle panels: VDOS of Fe(II) cyt c and Fe(III)
cyt c. Lower panel: VDOS of Fe(II), Fe(III) cyt c, and deoxyMb
represented in terms of the integrand D(ν j)ν j2of the mean Fe-ligand
bond stiffness (eq 5). The area determines the Fe-ligand bond stiffness.
J. Phys. Chem. B, Vol. 113, No. 7, 2009
Leu et al.
factors and multiphonon contributions to yield the normalized
one-phonon contribution S1′(ν j) to the NRVS signal and the Fe-
weighted vibrational density of states (VDOS)
which defines the vibrational properties at all temperatures for
a harmonic system.27
The sample temperatures are confirmed by requiring that the
ratio S′(ν j)/S′(-ν j) equal the Boltzmann factor exp(hcν j/kBT).
Measurements on a randomly oriented sample such as a solution
or polycrystalline powder yield the total VDOS D(ν j) summed
over the three Cartesian directions. Each mode R contributes
to D(ν j) an area ejR2, with j ) Fe, equal to the fraction of the
kinetic energy associated with motion of the iron atom.28The
total density of states is given by
Mode composition factors ejR2can be estimated from reported
frequency shifts in mode R resulting from small changes in the
mass of atom j according to the formula28
Equation 4 is generally applicable in the harmonic approxima-
tion, as long as the mode character is insensitive to mass
changes, and provides a useful method to estimate mode
character from isotopic frequency shifts.
3. Results and Discussion
3.1. Stiffness of the Fe Environment. Figure 2 (upper and
middle panels) presents the VDOS derived from NRVS data
recorded on reduced and oxidized cyt c. The dominant features
appear in the 300-400 cm-1region, a frequency range
comparable to other low-spin heme complexes36,46,47but much
higher than for high-spin complexes.33,47Inspection reveals that
this feature shifts down from 340-400 cm-1in reduced cyt c
to 320-380 cm-1in oxidized cyt c. Comparison with Raman
scattering measurements, in the following section, reveals
significant unresolved structure in this region.
However, we begin by considering the mean stiffness48,49of
the iron environment
as determined directly from the VDOS and reported in Table
1. (Here, ω ) 2πcν j.) The stiffness is the force constant for
displacement of the Fe nucleus along the direction of the X-ray
beam, with other nuclei fixed at their equilibrium positions. The
factor of 3 in eq 5 acknowledges an averaging over all directions
for the randomly oriented molecules in the frozen solutions
considered here. The stiffness can also be determined directly
from the measured excitation probability48if needed, without
invoking the assumption of harmonic behavior.
The quantity ν j2D(ν j) displayed in the lower panel of Figure 2
is proportional to the integrand of eq 5. These stiffness spectra
illustrate the dominant contribution of the 300-400 cm-1
frequency region to the stiffness of the Fe environment in cyt
c. The corresponding spectrum for deoxyMb is included for
comparison and reflects a dominant contribution centered near
250 cm-1, a smaller area, and a correspondingly lower stiffness
(Table 1). In contrast, frequencies below 100 cm-1largely
determine the resilience kr) mFe〈ω-2〉-1, a distinct force constant
that characterizes the magnitude of thermal fluctuations of the
Fe, as described in a separate publication.37
The nearest neighbors of the Fe atom can be expected to have
the primary influence on the stiffness, which thus probes the
local structure. This expectation implicitly motivates many
applications of vibrational spectroscopy, and a quantitative
inverse correlation between frequency and bond length50,51may
apply to vibrations localized on a single bond.52,53For example,
empirical valence bond parameters used to reproduce NRVS
data on rubredoxin featured a significantly decreased force
constant for stretching of the Fe-S bond in the reduced state,
consistent with a reported 5 pm increase in bond length.54We
anticipate that the stiffness may monitor changes in Fe-ligand
bond length even in complex situations where it is not possible
to assign vibrational frequencies to individual bonds and where
empirical approaches may not provide unique force constants.
Comparison of the stiffness with structural parameters derived
from EXAFS measurements17,55-57and summarized in Table 1
encourages this expectation. In particular, both the equatorial
Fe-Npyrbonds and the axial Fe-Nε(His) bond are significantly
longer in deoxyMb, in which the Fe is high spin, than in cyt c,
which has a low-spin Fe. The increased bond lengths correspond
to a large downshift of the dominant NRVS signal in deoxyMb
(Figure 2, lower panel) and a correspondingly reduced stiffness.
Reduced vibrational frequencies in the high-spin state are
commonly observed for iron complexes and contribute to the
entropy changes responsible for the temperature dependence of
The decreased stiffness observed upon oxidation of cyt c
(Table 1) is smaller, but still significant, reflecting more modest
structural changes. The Fe-S bond to Met80 exhibits the biggest
change upon oxidation17(Table 1). Other studies reveal some
quantitative variation in the absolute Fe-S bond length, with a
value as large as 241 pm reported for Fe(III) cyt c,56but the
qualitative increase upon oxidation appears to be repro-
ducible.17,56,57In contrast, smaller (1-2 pm) decreases reported
for the five remaining Fe-N bonds17are not consistently
reproduced in other studies56,57and may lie within the experi-
mental uncertainty. Thus, the reduced stiffness upon oxidation
of cyt c tracks small structural changes that challenge the
sensitivity of structural techniques, with the increased Fe-S
bond length likely to make the primary contribution. Chemical
criteria also suggest that the Fe-S(Met80) bond is stronger in
the reduced form, where it is more resistant to displacement by
exogenous ligands (ref 20 and references therein).
D(ν ¯) ) 3ν ¯
f [1 - exp(-hcν ¯
D(ν ¯) ) ∑ejR
2L(ν ¯ - ν ¯R)
∫D(ν ¯) dν ¯ ) 3
2) d(ln ν ¯R)/d(ln mj)
ks) mFe〈ω2〉 ) mFe1
TABLE 1: Average Fe-Ligand Bond Stiffness and Selected
Bond Lengths for Horse Heart Cyt c and DeoxyMb
Fe(II) cyt c
Fe(III) cyt c
stiffness (pN/pm)322 ( 17284 ( 17 190 ( 20
Vibrational Dynamics of Iron in Cytochrome cJ. Phys. Chem. B, Vol. 113, No. 7, 2009 2195
NRVS measurements on cyt f also found that the stiffness of
the low-spin Fe environment was significantly larger than in
high-spin forms of myoglobin.49However, stiffness differences
between oxidized and reduced states of cyt f were within the
experimental uncertainty. Although replacement of the Met
sulfur in cyt c with the nitrogen of the amino terminus as axial
ligand in cyt f might be expected to affect the Fe environment,
the slightly reduced stiffness of Fe(II) cyt c (Table 1) compared
to the 342 ( 18 pN/pm value reported for Fe(II) cyt f49is not
The stiffness spectra (Figure 2, lower panel) clearly identify
frequency regions that reflect the structure of the Fe environ-
ment. NRVS measurements on deoxyMb identified vibrations
of both axial and equatorial Fe-N bonds in the 230-260 cm-1
range.33For cyt c, vibrational markers for Fe-ligand bond
strength are most likely to appear in the 300-420 cm-1region.
Because cyt c NRVS signals appear to be more congested than
those previously reported, it is more challenging to resolve
features associated with individual vibrations.
Nevertheless, the stiffness responds to variations in Fe-
ligand bond length (Table 1) without the need to impose an
empirical model or to assign individual vibrations. Moreover,
comparison with higher resolution measurements on cyt c may
identify specific frequencies sensitive to the strength of indi-
3.2. Fe Vibrational Decomposition for Fe(II) Cyt c.
Comparison with isotope shift measurements provides more
detailed information on the vibrations contributing to the
stiffness. Figure 3 presents Raman spectra of reduced54Fe- and
57Fe-reconstituted cyt c, excited at a 413.1 nm wavelength.
Subtraction of these spectra reveals significant Fe isotope shifts
for numerous bands in the 300-420 cm-1region, while the
447, 479, 521, 538, 551, and 570 cm-1bands do not shift upon
57Fe f54Fe isotope substitution, consistent with the absence of
NRVS signal above 440 cm-1(Figure 2).
Figure 4 compares the Fe VDOS for Fe(II) cyt c (upper panel)
with the Raman spectra for57Fe- and54Fe-reconstituted Fe(II)
cyt c (middle and lower panels). The Raman band widths are
relatively narrow in comparison with typical heme proteins in
solution at room temperature, as noted previously for cyt c,43
which facilitates deconvolution into individual lines. We used
the Levenberg-Marquardt algorithm to fit the Raman spectra
in the 300-450 cm-1region with 10 Lorentzian bands, with
all fitting parameters free to vary. Table 2, columns 1-5, lists
their positions, line widths Γ, and the mode composition factors
eFe2determined from the isotope shifts according to eq 4.
Attempts to fit to Gaussians required additional bands to obtain
comparable fitting quality. Our frequencies for these 10 bands
for natural abundance Fe(II) cyt c (not listed) agree within 1
cm-1with other reported measurements.60,61
The mode composition factors estimated from the Raman
shifts according to eq 4 determine the expected contribution to
Figure 3. Raman spectra of57Fe and54Fe(II) cyt c and their spectral
difference demonstrate the presence of numerous Fe modes in the
Figure 4. VDOS of57Fe(II) cyt c (upper panel) and Raman spectra of
57Fe(II)- and54Fe(II)-reconstituted cyt c (lower panels). The error bars
represent the experimental statistical error in the NRVS experiment.
Dashed curves indicate components of a fit to the Raman data, with
the frequencies and mode composition factors compiled in Table 2.
Solid and dot-dash traces in the upper panel indicate contributions of
these modes to the Fe VDOS, predicted from the tabulated parameters,
assuming that either one or two modes, respectively, contribute to the
TABLE 2: Frequencies, Line Widths, and Mode
Composition Factors of the Raman Bands for Cyt ca
16 16 15
aThe iron mode composition factors were determined from eq 4.
J. Phys. Chem. B, Vol. 113, No. 7, 2009
Leu et al.
the VDOS. We use line widths ΓNRVS) ΓRaman+ 5.2 cm-1,
where ΓRamanare the line widths of the peaks in the Raman
spectrum (second column in Table 2) to account for the 5.2
cm-1difference between the resolutions of the two experiments.
Figure 4, upper panel, shows the predicted individual (Lorent-
zian) peaks and their sum. The total eFe2estimated for the peaks
listed in Table 2 is 1.33, accounting for 70% of the integrated
NRVS signal from 300 to 450 cm-1.
3.3. Vibrational Effects of Heme Oxidation. Previous
interpretations60-62have usually assumed a one-to-one cor-
respondence between Fe(II) and Fe(III) cyt c vibrational modes
in the 300-420 cm-1region, based on similarities between
Raman spectra in this region and their shifts due to isotope
substitutions.62We use the Raman spectra of the oxidized native
and57Fe-reconstituted reduced cyt c to test this assumption.
Figure 5 and Table 2, columns 1 and 6, indicate putative
correspondences60-62between the peaks in the reduced and
oxidized Raman spectra. We use the frequencies and line widths
determined from fitting the Raman data for native Fe(III) cyt c
(Figure 5, middle panel) and the eFe2values for Fe(II) cyt c
(Table 2) to predict the NRVS signal due to the Raman active
vibrations and compare it with the experimentally determined
VDOS (Figure 5, upper panel).
The comparison is not inconsistent with similar mode
character for most corresponding modes. The 375 cm-1mode
in Fe(III) cyt c is an exception, where the predicted NRVS signal
clearly exceeds the experimentally measured VDOS. Compari-
son of the results in the top panels of Figures 4 and 5 indicates
that the Fe amplitude of the 375 cm-1mode in Fe(III) cyt c is
reduced in comparison with the 372 cm-1mode in Fe(II) cyt c.
On the other hand, and in contrast with Fe(II) cyt c, the
maximum of the Fe(III) cyt c VDOS near 340 cm-1does not
correspond to any observed Raman mode.
3.4. Summary and Comparison with Other Heme Pro-
teins. NRVS and resonance Raman spectroscopy present
complementary solutions to the problem of spectral congestion,
which is central to vibrational studies of complex macromol-
ecules. The NRVS and Raman results presented here selectively
reveal vibrations of the heme active site of cyt c, free from
spectral interference from the surrounding polypeptide and
solvent. All vibrations involving Fe contribute to the VDOS
resulting from NRVS measurements. On the other hand, Raman
measurements provide higher spectral resolution at the present
time. Quantitative comparison of these two methods reveals a
more complete picture of the heme vibrations than is available
from either method individually.
For Fe(II) cyt c, we identify at least nine vibrational
frequencies in the 300-450 cm-1range that contribute to both
NRVS and Raman signals (Table 2). Raman measurements on
cyt c isotopomers resolve individual vibrational components of
the NRVS signal. These do not completely account for the
observed NRVS signal, which therefore contains contributions
from additional Fe vibrations, particularly near 340 cm-1.
All the Raman bands in the 300-420 cm-1region shift upon
isotope substitution (Figure 4), indicating that these modes
involve Fe motion. As a consequence, there is a large (70%)
Raman mode contribution to the NRVS signal above 300 cm-1.
A previous study on reduced cyt f49supplemented eight Raman
lines observed in this region with seven additional peaks in order
to fit the NRVS signal and also concluded that all Raman lines
involve iron motion. However, no Fe isotope shift data were
Raman measurements on myoglobin have occasionally identi-
fied vibrational frequencies sensitive to Fe isotope.34,63,64
Identification of the Fe-His frequency in the Raman spectrum
of deoxymyoglobin,63as well as a mode with Fe-NO stretching
character in MbNO,34was facilitated by observation of a single
Fe isotope-sensitive mode in the expected frequency region.
In contrast, essentially all modes in the 300-420 cm-1region
of the Raman spectrum of Fe(II) cyt c exhibit varying degrees
of sensitivity to the Fe isotope (Figures 3 and 4, Table 2). Fe-S
vibrations of Cys-ligated hemes have been reported in this
frequency region,65,66and mixing of axial ligand vibrations with
porphyrin vibrations might contribute to the observed vibrational
complexity. However, notice that Fe motion along one direction
accounts for only one-third of the Fe VDOS,33whose total area
is normalized to 3 (eq 3). Since ∑eFe2> 1 for the modes listed
in Table 2, Fe motion perpendicular to the axial bonds must
also contribute to both Raman and NRVS signals in this region
and may represent the primary contribution.
For a 4-fold D4h-symmetric Fe environment, in-plane Fe
modes occur as degenerate pairs with equal eFe2values and
orthogonal Fe motion, and they are Raman inactive in resonance
with the heme Soret band. However, difference measurements
reveal that in-plane Fe-Npyrvibrations contribute measurably
to the MbNO Raman signal.34Several factors reduce the nominal
4-fold symmetry of the heme. Splitting of the FeO tilting mode
in hydroxymetmyoglobin as a result of oxygen protonation35
demonstrates that asymmetry in the axial ligand can lift the
degeneracy of in-plane vibrations. However, frequency splittings
induced by heme asymmetry in cyt c may be too small to resolve
for some modes (see next section).
The asymmetric thioether linkages not only perturb the
approximate 4-fold symmetry of the heme but also induce
substantial distortion of the porphyrin from its nominal planar
Figure 5. Experimentally determined VDOS for Fe(III) cyt c (upper
panel) and Raman spectra of native Fe(III) and57Fe(II) cyt c (lower
panels). The error bars represent the experimental statistical error in
the NRVS experiment. Dashed curves in the upper panel were generated
from the band positions and line widths of native Fe(III) cyt c and the
isotope shifts for57Fe(II) cyt c (Table 2). Dashed curves in the middle
and lower panels are the peaks corresponding to data fitting with
Vibrational Dynamics of Iron in Cytochrome cJ. Phys. Chem. B, Vol. 113, No. 7, 2009 2197
geometry. The reduced symmetry may enable observation of
vibrational modes that would otherwise be forbidden by
selection rules and contribute to the increased vibrational
complexity. A complex vibrational signal is observed in the
same frequency region for cyt f,49although the detailed structure
differs, because of axial ligation with the nitrogen atom of the
terminal amino group rather than a methionine. Thus, we expect
that such vibrational complexity is probably a general feature
of thioether-linked hemes. On the other hand, nitrophorin67and
horseradish peroxidase68,69exhibit less vibrational complexity
in this region, in spite of hemes with significantly higher
distortion from planarity than myoglobin. As a result, we suggest
that the thioether links play a more significant role in reducing
the degree of vibrational symmetry than does the distortion of
3.5. Assessment of Previous Mode Assignments. Previous
Raman studies have suggested various assignments for specific
features in the 340-420 cm-1region. Early studies suggested
that some bands might be difference combinations70or overtones
or combinations and out-of-plane pyrrole tiltings.71We showed28,47
that the area contributed to the NRVS signal by an overtone or
a combination is smaller than the areas of the corresponding
fundamentals by a factor comparable to (ν j/ν jR)/eFe2, with ν jR)
15.8 cm-1for57Fe. The absence of intense features due to
vibrational fundamentals below 250 cm-1clearly rules out the
possibility that overtones and/or combinations contribute sig-
nificantly to the NRVS signal in the 320-420 cm-1region. We
conclude that all frequencies in Table 2 correspond to vibrational
The 307 and 358 cm-1bands were previously assigned as
degenerate pairs of modes (ν51and ν50, respectively),62involving
asymmetric stretching of the Fe-Npyrbonds along two orthogo-
nal in-plane directions. To test this idea, we double the area of
the 307 cm-1peak. The new prediction matches the Fe VDOS
better (Figure 4, dash-and-dot line in the upper panel). Therefore,
our results are consistent with the assignment of the 307 cm-1
band to a degenerate pair of modes, possibly with a ν51character.
By doubling the predicted area of the 358 cm-1peak,
agreement with the NRVS signal is not significantly improved
or worsened. This analysis is not inconsistent with assignment
as a degenerate mode pair with ν50character.62However, we
cannot exclude assignment to a single mode, possibly involving
stretching of the axial Fe-N and Fe-S bonds.72
The relatively weak sensitivity of the 348 cm-1mode to Fe
isotope substitution (eFe2) 0.06, Table 2) is consistent with its
assignment to the ν8 vibration,62which involves symmetric
stretching of the four Fe-Npyrbonds to the heme and thus leaves
the Fe atom motionless in 4-fold symmetric molecules.73This
disagrees with the cyt f study mentioned above,49which
concluded that involvement of Fe motion in this mode makes
the ν8label meaningless. Although there may be a small amount
of mixing with out-of-plane vibrations, the Fe amplitude remains
small, and we conclude that the ν8label remains meaningful
for cyt c.
The 392, 401, 414, and 422 cm-1bands are sensitive to N,
Ca, and Cbisotope substitutions (Figure 1) and were assigned
as (C?-Ca-Cb) and (C?-Ca-S) bending modes of the thioether
groups.62Because of the complex character of these modes
(involving motions of both the pyrrole nitrogens and the
peripheral groups), it is not unexpected that they involve Fe
motion as well. Indeed, we find significant eFe2values for these
modes (Table 2).
In the same work,62the 372 and 382 cm-1bands were
assigned as propionate bending modes (δ(C?-Cc-Cd)). They
are nearly insensitive to N, Ca, and Cbisotope substitution, and
the assignment was made by analogy with myoglobin, where
modes at nearby frequencies were assigned on the basis of
isotope shift upon propionate deuteration.74The 382 cm-1band
involves a relatively small iron motion, not inconsistent with
substantial contributions from propionate vibrations. However,
we find that the 372 cm-1band displays the biggest shift upon
57Fe f54Fe substitution and, consequently, the largest mode
composition factor eFe2(Table 2). This frequency corresponds
to the maximum NRVS signal, thus making substantial contri-
bution from propionate vibrations unlikely.
3.6. Identification of Structural Markers. The 320-420
cm-1region of the Soret-enhanced Raman spectrum of cyt c is
significantly richer than the corresponding region of proteins
such as myoglobin, which lack the thioether links to the protein.
This region is highly sensitive to protein conformation and has
been a useful fingerprint for identifying structural changes of
the heme environment upon mutation,21binding to ions71or lipid
membranes,22,75or unfolding.22,23In particular, changes in axial
ligation lead to spectral perturbations in this region.22,24,71,76,77
However, there are no generally accepted assignments of
observed Raman frequencies to vibrations of individual Fe-
ligand bonds in cyt c.
Although one might consider the possibility that such
structurally sensitive modes are not enhanced in resonance with
the Soret transition of the heme, the results presented here
suggest that this is not the case. Observed Raman modes account
for a large fraction of the NRVS signal above 300 cm-1, at
least for Fe(II) cyt c, and these vibrations make the primary
contribution to the stiffness of the Fe environment in both
oxidation states (Figure 2, Table 1).
One specific possibility that should be considered in future
investigations is that the 372 cm-1mode in Fe(II) cyt c
corresponds to the Fe-S stretching vibration, which may shift
down to ∼340 cm-1in Fe(III) cyt c and no longer contribute
to the Raman signal excited in resonance with the Soret
transition. The frequency shift would be consistent with the
modest increase in Fe-S bond length reported for the oxidized
protein17(Table 1) and would contribute to the reduced stiffness
of the oxidized protein. This frequency range is consistent with
previous observations of Fe(III)-S stretching vibrations near
350 cm-1in cyt P45065and chloroperoxidase.66On the basis of
a 1.5 cm-1shift upon56Fe f54Fe isotope substitution, the Fe-S
band was assigned at 328 cm-1in [Fe(TPP)(S(CH3)2]ClO4.78
The absence of a significant frequency shift upon deuteration
of the methionine methyl group,62together with the large eFe2
(Table 2), might indicate that the 372 cm-1vibration is relatively
localized on the iron and sulfur atoms. The total eFe2) 0.47
for this mode exceeds the value eFe2) mS/(mFe+ mS) ) 0.36
expected for a two-body Fe-S oscillator. Significant vibrational
alterations have been observed in this frequency range upon
replacement of the methionine sulfur ligand with the nitrogen
atom of the protein amino terminus in cyt f.49,79The maximum
of the Fe VDOS decreases considerably upon oxidation (Figure
2), consistent with the longer Fe-S(Met80) bond in oxidized
cyt c (Table 1). We suggest that the 372 cm-1band is the
Fe-S(Met80) stretching mode in reduced cyt c. This band does
not have an obvious correspondent in the Raman spectrum of
oxidized cyt c.
The doubling of the area of the 358 cm-1peak has a minimal
effect on the predicted NRVS signal, which may indicate that
it has an out-of-plane character. This mode was previously
assigned as a stretching of the Fe-N and Fe-S bonds.72Its
mode composition factor eFe2) 0.14 lies within a range of
J. Phys. Chem. B, Vol. 113, No. 7, 2009
Leu et al.
values 0.11-0.18 calculated for a two-body oscillator, assuming
various distributions of Fe, Met, and His masses. Given the
relatively large value of the iron mode composition factor, a
NRVS measurement on an15N(His) and/or34S(Met) isotopically
labeled cyt c might clarify the assignment of this mode.
Vibrations of the Fe-N bond to the histidine also must
contribute to the NRVS signal. Although Fe-histidine vibrations
are not observed to contribute significantly to the Raman signal
for six-coordinated heme proteins, five-coordinated heme pro-
teins exhibit strongly enhanced Fe-His vibrations in the
200-250 cm-1region.63,80As a result, we expect that the
Fe-His vibration may contribute to one or more of the features
observed in the 100-300 cm-1range of the VDOS (Figure 2).
A band between 176 and 183 cm-1in the resonance Raman
spectra of several mitochondrial cyt c was assigned as a
ν(Fe-His) mode coupled to a ν(Fe-S(Met)) mode.81,82
However, we acknowledge the possibility that vibrations may
resist simple description in terms of motion localized to
individual bonds. In a thorough study of low-spin iron porphy-
rins with imidazole and CO ligands, we identified several
vibrations involving both Fe and imidazole.36Hence, it may
not always be possible to identify a single feature with vibration
of each Fe-ligand bond. Naturally occurring porphyrins have
a rich vibrational structure, and comparison with quantum
chemical calculations may provide valuable guidance for
establishing the sensitivity of vibrational features to the strength
of particular Fe-ligand bonds.36,46,47,83-86
As for other57Fe-containing proteins,29,33,34,49,87,88,54NRVS
provides a site-selective view of active site vibrations in cyt c
that complements information available from well-established
vibrational techniques such as resonance Raman spectroscopy.
NRVS reveals the complete VDOS of the Fe atom, allowing
evaluation of effective force constants. The stiffness measures
the force required to displace the Fe relative to its nearest
neighbors and decreases slightly upon oxidation of cyt c (Table
1).48This contrasts with the resilience, which is related to the
mean-squared displacement of the Fe atom in response to
thermal fluctuations.37The resilience is relatively insensitive to
the oxidation state of cyt c, and the decreased resilience observed
for myoglobin may reflect the absence of the thioether bonds.37
In contrast, the stiffness of cyt c decreases slightly upon
oxidation, reflecting the weakened Fe-S bond.
Comparison with Raman measurements on cyt c isotopically
labeled at the Fe enables a more detailed description of the
vibrational structure. Previous NRVS studies of heme proteins
have revealed Fe-ligand modes not reported in resonance
Raman spectra, such as the Fe-Npyrbonds to the heme pyrrole
ligands.33For Fe(II) cyt c, however, we find that vibrations
observed in the Raman spectrum account for a significant
fraction of the NRVS signal above 300 cm-1. This indicates
that both axial and equatorial Fe ligand vibrations contribute to
the Raman signal. We suggest that observation of many of these
modes is forbidden by selection rules in proteins with more
symmetric heme groups. Both the asymmetrically placed
thioether links and the resulting heme nonplanarity may
contribute to allow these modes to couple to the electronic Soret
transitions of the heme in cyt c.
The rich vibrational dynamics of Fe reported here for cyt c
form an intriguing comparison with earlier measurements on
Mb. For deoxyMb,57Fe NRVS data resolved three primary
features that we associated with stretching of axial and equatorial
Fe-N bonds,33and comparison with DFT predictions supports
a similarly simple interpretation of high-frequency Fe vibrations
in ferryl Mb.35In contrast, more than 10 vibrational modes
contribute to determining the stiffness of the Fe environment
in Fe(II) cyt c, although there are only three chemically distinct
bond types to the heme Fe. Further investigation should enable
the use of this rich vibrational information to monitor confor-
mational changes of cyt c upon complex formation with
phospholipid membranes, as proposed to take place within
mitochondria75or during cellular apoptosis.6
Acknowledgment. We thank Prof. Abel Schejter for useful
discussions and acknowledge generous support of this research
by the National Science Foundation (PHY-0545787). Use of
the Advanced Photon Source was supported by the U.S.
Department of Energy, Basic Energy Sciences, Office of
Science, under Contract DE-AC02-06CH11357.
References and Notes
(1) Berg, J.; Tymoczko, J.; Stryer, L. Biochemistry, 5th ed.; Freeman
and Co.: New York, 2002.
(2) Scott, R. A.; A. G. Mauk, E. Cytochrome c: A Multidisciplinary
Approach; University Science Books: Sausalito, 1996.
(3) Moore, G. R.; Pettigrew, G. W. Cytochromes c: Biological Aspects;
Springer-Verlag: New York, 1987.
(4) Moore, G. R.; Pettigrew, G. W. Cytochromes c: EVolutionary,
Structural, and Physicochemical Aspects; Springer-Verlag: New York, 1990.
(5) Jiang, X.; Wang, X. Annu. ReV. Biochem. 2004, 73, 87–106.
(6) Bayir, H.; Fadeel, B.; Palladino, M. J.; Witasp, E.; Kurnikov, I. V.;
Tyurina, Y. Y.; Tyurin, V. A.; Amoscato, A. A.; Jiang, J.; Kochanek, P. M.;
DeKosky, S. T.; Greenberger, J. S.; Shvedova, A. A.; Kagan, V. E. Biochim.
Biophys. Acta 2006, 1757, 648–659.
(7) Banci, L.; Bertini, I.; Gray, H. B.; Luchinat, C.; Reddig, T.; Rosato,
A.; Turano, P. Biochemistry 1997, 36, 9867–9877.
(8) Banci, L.; Bertini, I.; Huber, J. G.; Spyroulias, G. A.; Turano, P.
J. Biol. Inorg. Chem. 1999, 4, 21–31.
(9) Feng, Y.; Roder, H.; Englander, S. W. Biochemistry 1990, 29, 3494–
(10) Gao, Y.; Boyd, J.; Pielak, G. J.; Williams, R. J. P. Biochemistry
1991, 30, 1928–1934.
(11) Qi, P. X.; Beckman, R. A.; Wand, A. J. Biochemistry 1996, 35,
(12) Louie, G. V.; Brayer, G. D. J. Mol. Biol. 1990, 214, 527–555.
(13) Berghuis, A. M.; Brayer, G. D. J. Mol. Biol. 1992, 223, 959–976.
(14) Takano, T.; Dickerson, R. E. J. Mol. Biol. 1981, 153, 95–115.
(15) Takano, T.; Dickerson, R. E. J. Mol. Biol. 1981, 153, 79–94.
(16) Sanishvili, R.; Volz, K.; Westbrook, E.; Margoliash, E. Structure
1995, 3, 707–16.
(17) Cheng, M.-C.; Rich, A. M.; Armstrong, R. S.; Ellis, P. J.; Lay,
P. A. Inorg. Chem. 1999, 38, 5703–5708.
(18) Trewhella, J.; Carlson, V. A. P.; Curtis, E. H.; Heidorn, D. B.
Biochemistry 1988, 27, 1121–1125.
(19) Tiede, D. M.; Zhang, R.; Seifert, S. Biochemistry 2002, 41, 6605–
(20) Schejter, A. Cytochrome c: A Multidisciplinary Approach; Uni-
versity Science Books: Sausalito, CA, 1996; Chapter 8, p 335.
(21) Hildebrandt, P.; Pielak, G. J.; Williams, R. J. P. Eur. J. Biochem.
1991, 201, 211–216.
(22) Droghetti, E.; Oellerich, S.; Hildebrandt, P.; Smulevich, G. Biophys.
J. 2006, 91, 3022–3031.
(23) Takahashi, S.; Yeh, S.-R.; Das, T. K.; Chan, C.-K.; Gottfried, D. S.;
Rousseau, D. L. Nat. Struct. Biol. 1997, 4, 44–50.
(24) Yeh, S.-R.; Rousseau, D. L. J. Biol. Chem. 1999, 274, 17853–
(25) Sturhahn, W.; Toellner, T. S.; Alp, E. E.; Zhang, X.; Ando, M.;
Yoda, Y.; Kikuta, S.; Seto, M.; Kimball, C. W.; Dabrowski, B. Phys. ReV.
Lett. 1995, 74, 3832–3835.
(26) Zeng, W.; Silvernail, N. J.; Scheidt, W. R.; Sage, J. T. In
Applications of Physical Methods to Inorganic and Bioinorganic Chemistry;
Scott, R. A., Lukehart, C. M., Eds.; John Wiley & Sons, Ltd.: Chichester,
UK, 2007; pp 401-421.
(27) Sturhahn, W. J. Phys.: Condens. Matter 2004, 16, S497–S530.
(28) Sage, J. T.; Paxson, C.; Wyllie, G. R. A.; Sturhahn, W.; Durbin,
S. M.; Champion, P. M.; Alp, E. E.; Scheidt, W. R. J. Phys.: Condens.
Matter 2001, 13, 7707–7722.
(29) Bergmann, U.; Sturhahn, W.; Linn, D. E.; Jenney, F. E.; Adams,
M. W.; Rupnik, K.; Hales, B. J.; Alp, E. E.; Mayse, A.; Cramer, S. P.
J. Am. Chem. Soc. 2003, 125, 4016–4017.
Vibrational Dynamics of Iron in Cytochrome c J. Phys. Chem. B, Vol. 113, No. 7, 2009 2199
(30) Chumakov, A. I.; Sergueev, I.; van Bu ¨rck, U.; Schirmacher, W.;
Asthaller, T.; Ru ¨ffer, R.; Leupold, O.; Petry, W. Phys. ReV. Lett. 2004, 92,
(31) Tse, J.; Klug, D.; Zhao, J.; Sturhahn, W.; Alp, E.; Baumert, J.;
Gutt, C.; Johnson, M.; Press, W. Nat. Mater. 2005, 4, 917–921.
(32) Giefers, H.; Tanis, E. A.; Rudin, S. P.; Greeff, C.; Ke, X.; Chen,
C. F.; Nicol, M. F.; Pravica, M.; Pravica, W.; Zhao, J.; Alatas, A.; Lerche,
M.; Sturhahn, W.; Alp, E. Phys. ReV. Lett. 2007, 98, 245502.
(33) Sage, J. T.; Durbin, S. M.; Sturhahn, W.; Wharton, D. C.;
Champion, P. M.; Hession, P.; Sutter, J.; Alp, E. E. Phys. ReV. Lett. 2001,
(34) Zeng, W.; Silvernail, N. J.; Wharton, D. C.; Georgiev, G. Y.; Leu,
B. M.; Scheidt, W. R.; Sturhahn, W.; Alp, E. E.; Sage, J. T. J. Am. Chem.
Soc. 2005, 127, 11200–11201.
(35) Zeng, W.; Barabanschikov, A.; Zhang, Y.; Zhao, J.; Sturhahn, W.;
Alp, E. E.; Sage, J. T. J. Am. Chem. Soc. 2008, 130, 1816–1817.
(36) Leu, B. M.; Silvernail, N. J.; Zgierski, M. Z.; Wyllie, G. R. A.;
Ellison, M. K.; Scheidt, W. R.; Zhao, J.; Sturhahn, W.; Alp, E. E.; Sage,
J. T. Biophys. J. 2007, 92, 3764–3783.
(37) Leu, B. M.; Zhang, Y.; Bu, L.; Straub, J. E.; Zhao, J.; Sturhahn,
W.; Alp, E. E.; Sage, J. T. Biophys. J. 2008, 95, 5874–5889.
(38) Morell, D. B.; Stewart, M. Aust. J. Exp. Biol. Med. Sci. 1956, 34,
(39) Vanderkooi, J. M.; Adar, F.; Erecinska, M. Eur. J. Biochem. 1976,
(40) Dickinson, L. C.; Chien, J. C. W. Biochemistry 1975, 14, 3526–
(41) Vanderkooi, J. M.; Landesburg, R.; Hayden, G. W.; Owen, C. S.
Eur. J. Biochem. 1977, 81, 339–347.
(42) Leu, B. M. Nuclear resonance vibrational spectroscopy: A quantita-
tive picture of iron dynamics in heme proteins and model compounds. PhD
Thesis, Northeastern University, 2006.
(43) Schomacker, K. T.; Champion, P. M. J. Chem. Phys. 1989, 90,
(44) Seto, M.; Yoda, Y.; Kikuta, S.; Zhang, X. W.; Ando, M. Phys.
ReV. Lett. 1995, 74, 3828–3831.
(45) Sturhahn, W. Hyperfine Interact. 2000, 125, 149–172.
(46) Leu, B. M.; Zgierski, M. Z.; Wyllie, G. R. A.; Scheidt, W. R.;
Sturhahn, W.; Alp, E. E.; Durbin, S. M.; Sage, J. T. J. Am. Chem. Soc.
2004, 126, 4211–4227.
(47) Leu, B. M.; Zgierski, M. Z.; Wyllie, G. R. A.; Ellison, M. K.;
Scheidt, W. R.; Sturhahn, W.; Alp, E. E.; Durbin, S. M.; Sage, J. T. J.
Phys. Chem. Solids 2005, 66, 2250–2256.
(48) Lipkin, H. J. Phys. ReV. B 1995, 52, 10073–10079.
(49) Adams, K. L.; Tsoi, S.; Yan, J.; Durbin, S. M.; Ramdas, A. K.;
Cramer, W. A.; Sturhahn, W.; Alp, E. E.; Schulz, C. J. Phys. Chem. B
2006, 110, 530–536.
(50) Badger, R. M. J. Chem. Phys. 1935, 3, 710–714.
(51) Dyer, R. B.; Woodruff, W. H. In Applications of Physical Methods
to Inorganic and Bioinorganic Chemistry; Scott, R. A., Lukehart, C. M.,
Eds.; John Wiley & Sons, Ltd.: Chichester, UK, 2007; pp 001-024.
(52) Green, M. T. J. Am. Chem. Soc. 2006, 128, 1902–1906.
(53) Xu, C.; Spiro, T. G. J. Biol. Inorg. Chem. 2008, 13, 613–621.
(54) Xiao, Y.; Wang, H.; George, S. J.; Smith, M. C.; Adams, M. W. W.;
Jenney, F. E.; Sturhahn, W.; Alp, E. E.; Zhao, J.; Yoda, Y.; Dey, A.;
Solomon, E. I.; Cramer, S. P. J. Am. Chem. Soc. 2005, 127, 14596–14606.
(55) Chance, M. R.; Miller, L. M.; Fischetti, R. F.; Scheuring, E.; Huang,
W.-X.; Sclavi, B.; Hai, Y.; Sullivan, M. Biochemistry 1996, 35, 9014–
(56) Hsu, I.-J.; Shiu, Y.-J.; Jeng, U.-S.; Chen, T.-H.; Huang, Y.-S.; Lai,
Y.-H.; Tsai, L.-N.; Jang, L.-Y.; Lee, J.-F.; Lin, L.-J.; Lin, S.-H.; Wang, Y.
J. Phys. Chem. A 2007, 111, 9286–9290.
(57) Giachini, L.; Francia, F.; Cordone, L.; Boscherini, F.; Venturoli,
G. Biophys. J. 2007, 92, 1350–1360.
(58) Brehm, G.; Reiher, M.; Schneider, S. J. Phys. Chem. A 2002, 106,
(59) Ronayne, K. L.; Paulsen, H.; Hofer, A.; Dennis, A. C.; Wolny,
J. A.; Chumakov, A. I.; Schunemann, V.; Winkler, H.; Spiering, H.;
Bousseksou, A.; Gutlich, P.; Trautwein, A. X.; McGarvey, J. J. Phys. Chem.
Chem. Phys. 2008, 8, 4685–4693.
(60) Hildebrandt, P. Cytochrome c: A Multidisciplinary Approach;
University Science Books: Sausalito, CA, 1996; pp 285-314.
(61) Schomacker, K. Absorption, resonant Rayleigh and resonance
Raman properties of cytochrome c. PhD Dissertation, Department of Physics,
Northeastern University, 1987.
(62) Hu, S.; Morris, I. K.; Singh, J. P.; Smith, K. M.; Spiro, T. G. J. Am.
Chem. Soc. 1993, 115, 12446–12458.
(63) Argade, P. V.; Sassaroli, M.; Rousseau, D. L.; Inubushi, T.; Ikeda-
Saito, M.; Lapidot, A. J. Am. Chem. Soc. 1984, 106, 6593–6596.
(64) Hirota, S.; Mizoguchi, Y.; Yamauchi, O.; Kitagawa, T. J. Biol.
Inorg. Chem. 2002, 7, 217–221.
(65) Champion, P. M.; Stallard, B.; Wagner, G. C.; Gunsalus, I. C. J. Am.
Chem. Soc. 1982, 104, 5469–5472.
(66) Bangcharoenpaurpong, O.; Champion, P. M.; Hall, K.; Hager, L. P.
Biochemistry 1986, 25, 2374–2378.
(67) Maes, E. M.; Walker, F. A.; Montfort, W. R.; Czernuszewicz, R. S.
J. Am. Chem. Soc. 2001, 123, 11664–11672.
(68) Heering, A.; Smith, A. T.; Smulevich, G. Biochem. J. 2002, 363,
(69) Gruia, F.; Kubo, M.; Ye, X.; Ionascu, D.; Lu, C.; Poole, R. K.;
Yeh, S.-R.; Champion, P. M. J. Am. Chem. Soc. 2008, 130, 5231–5244.
(70) Friedman, J. M.; Hochstrasser, R. M. Chem. Phys. 1973, 1, 457–
(71) Hildebrandt, P. Biochim. Biophys. Acta 1990, 1040, 175–186.
(72) Hon-Nami, K.; Kihara, H.; Kitagawa, T.; Miyazawa, T.; Oshima,
T. Eur. J. Biochem. 1980, 110, 217–223.
(73) Abe, M.; Kitagawa, T.; Kyogoku, Y. J. Chem. Phys. 1978, 69,
(74) Hu, S.; Smith, K. M.; Spiro, T. G. J. Am. Chem. Soc. 1996, 118,
(75) Berezhna, S.; Wohlrab, H.; Champion, P. M. Biochemistry 2003,
(76) Dopner, S.; Hildebrandt, P.; Rosell, F. I.; Mauk, A. G. J. Am. Chem.
Soc. 1998, 120, 11246–11255.
(77) Hirota, S.; Suzuki, M.; Watanabe, Y. Biochem. Biophys. Res.
Commun. 2004, 314, 452–458.
(78) Oshio, H.; Ama, T.; Watanabe, T.; Nakamoto, K. Inorg. Chim. Acta
1985, 96, 61–66.
(79) Desbois, A. Biochimie 1994, 76, 693–707.
(80) Wells, A. V.; Sage, J. T.; Morikis, D.; Champion, P. M.; Chiu,
M. L.; Sligar, S. G. J. Am. Chem. Soc. 1991, 113, 9655–9660.
(81) Cartling, B. Biophys. J. 1983, 43, 191–205.
(82) Desbois, A.; Lutz, M. Proceedings of the XIth International
Conference on Raman Spectroscopy, Tokyo; The Chemical Society of Japan:
Tokyo, 1984; pp 480-481.
(83) Vogel, K. M.; Kozlowski, P. M.; Zgierski, M. Z.; Spiro, T. G. J. Am.
Chem. Soc. 1999, 121, 9915–9921.
(84) Kozlowski, P. M.; Spiro, T. G.; Zgierski, M. Z. J. Phys. Chem. B
2000, 104, 10659–10666.
(85) Rush, T. S., III.; Kozlowski, P. M.; Piffat, C. A.; Kumble, R.;
Zgierski, M. Z.; Spiro, T. G. J. Phys. Chem. B 2000, 104, 5020–5034.
(86) Paulat, F.; Praneeth, V. K. K.; Nather, C.; Lehnert, N. Inorg. Chem.
2006, 45, 2835–2856.
(87) Keppler, C.; Achterhold, K.; Ostermann, A.; van Bu ¨rck, U.; Potzel,
W.; Chumakov, A. I.; Baron, A. Q.; Ru ¨ffer, R.; Parak, F. Eur. Biophys. J.
1997, 25, 221–224.
(88) Achterhold, K.; Keppler, C.; Ostermann, A.; van Bu ¨rck, U.;
Sturhahn, W.; Alp, E. E.; Parak, F. G. Phys. ReV. E 2002, 65, 051916.
(89) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.;
Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28,
(90) Bushnell, G. W.; Louie, G. V.; Brayer, G. D. J. Mol. Biol. 1990,
(91) Sayle, R.; Milner-White, E. J. Trends Biochem. Sci. 1995, 20, 374.
(92) Portman, S.; Lu ¨thi, H. P. Chimia 2000, 54, 766–770.
J. Phys. Chem. B, Vol. 113, No. 7, 2009
Leu et al.