Jung-2000-Journal of Molecular Recognition

JOURNAL OF MOLECULAR RECOGNITION
J. Mol. Recognit. 2000;13:325351
Review
Insight into protein structure and proteinligand

recognition by Fourier transform infrared
spectroscopy
Christiane Jung*
Max-Delbruck-Center for Molecular Medicine, D-13125 Berlin, Germany
An overview of the application of Fourier transform infrared spectroscopy for the analysis of the structure
of proteins and proteinligand recognition is given. The principle of the technique and of the spectra
analysis is demonstrated. Spectral signal assignments to vibrational modes of the peptide chromophore,
amino acid side chains, cofactors and metal ligands are summarized. Several examples for proteinligand
recognition are discussed. A particular focus is heme proteins and, as an example, studies of cytochrome
P450 are reviewed. Fourier transform infrared spectroscopy in combination with the various techniques
such as time-resolved and low-temperature methods, site-directed mutagenesis and isotope labeling is a
helpful approach to studying proteinligand recognition. Copyright # 2000 John Wiley & Sons, Ltd.
Keywords: Fourier transform infrared spectroscopy; proteins; proteinligand recognition; heme proteins; cytochrome
P450
Received 9 June 2000; accepted 9 June 2000
INTRODUCTION
Molecular recognition is a very complex phenomenon.
Proteins, substrates and ligands are part of an ensemble of
various components of the solution or the cell which all
together contribute to the recognition process. How can one
figure out and separate all the different interactions and find
the interaction that is most relevant for the function? There
are many experimental and theoretical methods to approach
the problem. One of them is Fourier transform infrared
spectroscopy (FTIR). The application of FTIR spectroscopy
to proteins is a broad field. This review will focus on
selected topics which are connected with proteinligand
recognition and will take cytochrome P450 (P450) as the
primary example. P450s represent a big superfamily of
heme-type monooxygenases which catalyze the conversion
of diverse substrates (Lewis, 1996). In contrast to most
heme proteins, like myoglobin and hemoglobin, P450s face
the problem of different recognition phenomena. How the

protein recognizes the dioxygen (and other related small
heme iron ligands) and how it depends on the substrate
bound in the heme pocket are important questions for
uncovering the catalyzing function of this class of enzymes.
Interpretation of the infrared spectra of P450 benefits
significantly from the knowledge of infrared spectroscopy
on many other proteins which has been accumulated in the
literature over more than three decades. This will be briefly
reviewed and recognition phenomena, in particular in heme
proteins, will be discussed. Because there are so many
papers about FTIR studies, only the more recent ones will be
refered to the particular subject.
METHODOLOGY OF FOURIER
TRANSFORM INFRARED
SPECTROSCOPY
Instrumental principle
* Correspondence to: C. Jung, Max-Delbruck-Center for Molecular Medicine,

Research Group Protein Dynamics, Robert-Rossle-Strasse 10, D-13125
Berlin, Germany.
E-mail: cjung@mdc-berlin.de
Contract/grant sponsor: Deutsche Forschungsgemeinschaft; contract/grant
number: Ju229/1-1; contract/grant number: Ju229/3-1; contract/grant
number: Sk35/3-1,2.
Contract/grant sponsor: European Commission; contract/grant number:
BIO2-CT942060.
Abbreviations used: Adx, adrenodoxin; ATP, adenosine 5'-triphosphate; CD,
circular dichroism; DHAP, dihydroxyacetone phosphate; FTIR, Fourier
transform infrared spectroscopy; GAP, D-glyceraldehyde 3-phosphate; Pdx,
putidaredoxin; P450, cytochrome P450; P450cam, P450 from camphorhydroxylating Pseudomonas putida.
Copyright # 2000 John Wiley & Sons, Ltd.
In contrast to the dispersive spectrometer, where the

radiation from an infrared source is made monochromatic,
the Fourier transform spectrometer uses polychromatic
radiation. The collimated radiation from the infrared light
source is partially reflected and partially transmitted by the
beamsplitter (BS) [Fig. 1(A)]. The beam originally
transmitting the beamsplitter is then reflected from a fixed
mirror (FM) while the beam originally reflected from the
beamsplitter is reflected from a movable mirror (MM). Both
mirror-reflected beams are recombined and split again at the
beamsplitter, where one of the newly split beams goes back
326
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Figure 1. Principle of the experimental set-up of Fourier transform infrared

spectroscopy coupled with various applications of time-resolved, lowtemperature and high-pressure methods. (A) Right upper corner: interferometer with FM = xed mirror; MM = movable mirror; BS = beam splitter;
x = distance MM moves (scanner drive); MCT = HgCdTe detector; A/D = analogdigital converter; PC = personal computer; NdYAG = laser for photoinitiated processes, 1064 nm or 532 nm; M = mirror; ir-source = infrared light
source (globar). (B) Interferogram and (C) time trace for the rebinding of the
photodissociated CO ligand in (1R)-camphor-bound cytochrome P450cam
monitored at a specic MM mirror position (170 Dx increments apart from the
center burst); 50 mM potassium phosphate D2O buffer, pD 7; 50% (v/v)
glycerol-d3, c(P450) = 0.79 mM, 23.7 mM (1R)-camphor (Contzen and Jung,
1998).
to the source while the other one passes the sample and
reaches the detector. The recombination of the mirrorreflected beams leads to a complex interference pattern with
the highest intensity in the center burst and decreasing
intensity to the sides of the center with increasing or
decreasing distance (x) between the movable mirror and the
beamsplitter. The detector measures the intensity [I(x)] of
the resulting radiation as a function of the distance (x)
(interferogram) [Fig. 1(B)].
A calibrating beam of a HeNe laser goes the same path
as the infrared radiation. Because of the monochromatic
HeNe laser light, its interferogram is a sine function. The
distance between two zero crossing points is simply half the
wavelength l of the laser light which is used as an internal
frequency calibration. The relation between the intensity
I(x) at the mirror position x and the intensity of a
monochromatic spectral line S(n) at the wavenumber n (=
1/l) is given by eq. (1).
Ix S cos2x
The result of the data acquisition is a digitized interferogram

I(nDx), where n is the counting number of the distance
difference Dx between two zero crossing points of the He
Ne laser light. This interferogram is Fourier transformed

into the spectrum S(kDn) [eq. (2)] with k used as a counting
number of the frequency increment Dn. N is the total number
of digitized points.
Sk
N
1
X
Inx expi2nk=N
n0
1=N x
S(kDn) represents the so-called single channel spectrum
[Fig. 2(A)]. The ratio between two single channel spectra
(Ssample/Sreference) gives the transmission spectrum which is
transformed to the absorption spectrum [Fig. 2(B)] by taking
the negative decadic logarithm.
FT infrared spectroscopy has various advantages compared to the dispersive method: (i) absorbance differences of
only 104 at an absolute absorbance of 0.51 can clearly be
resolved with few scans; (ii) the measurement time is very
short a time resolution of several milliseconds with a
mirror velocity of 320 kHz and a resolution of 8 cm1 can
be realized in the rapid-scan mode; and (iii) the step-scan
technique of moving the mirror step-wise opens new
J. Mol. Recognit. 2000;13:325351
FTIR AND PROTEINLIGAND RECOGNITION
327
Time-resolved experiments are triggered or initiated by

various methods. Most of the studies are done with
flashphotolysis where a pulsed laser (for example the Nd
YAG laser) is used. The laser flash leads to the dissociation
of the ligand, for example from the heme iron or a functional
important agent (for example ATP) is released from a cage
(Cepus et al., 1998a,b). Light-induced structural changes
connected with protonation equilibria of the different
intermediates in the photocycle of bacteriorhodopsin has
been extensively studied by time-resolved FTIR (Rammelsberg et al., 1997; Rodig and Siebert, 1999). Temperature
jump experiments, in which a fast heating of the sample is
initiated by a laser (Phillips et al., 1995) or by a fast mixing
with a hot solution (Backmann et al., 1995) have been
reported to study protein dynamics and protein unfolding/
folding phenomena. The pressure jump (or release) technique has also been combined with FTIR (Frauenfelder et
al., 1990).
FTIR studies at low temperatures down to 10K are
usually performed with closed cycle helium cryosystems or
bath cryostates. High-temperature studies (up to 100C) are
commonly applied to study protein unfolding. Protein
unfolding is also studied extensively using high-pressure
anvil cells (Wong, 1987; Heremans, 1997). Vibrational
circular dichroism is one of the newest developments and
gives detailed structural information about molecules with
chiral properties. For details see the recent review by
Keiderling (1996). Extensively used is also the ATR
(attenuated total reflection) technique which allows the
study of the structure of proteins adsorbed on solid surfaces
or in lipids (Menestrina et al., 1999).
Spectra analysis
Figure 2. Infrared spectrum of substrate-free cytochrome
P450cam-CO. (A) Single-channel intensity spectra of the protein
sample (Ssample) in 100 mM potassium phosphate D2O buffer, pD
7, 20C (Mouro et al., 1997), and reference buffer (Sreference). (B)
Top: absorption spectrum obtained from the single channel
spectra in (A), log(Ssample/Sreference); middle: water spectra with
0% H2O, 25% H2O and 100% H2O taken with CaF2 windows and a
5 m teon spacer; and (C) water vapor spectrum with a small
impurity of CO2.
applications for time-resolved measurements. At each

mirror position, a chemical reaction or conformational
change is induced, for example by a laser flash. The time
evolution of the intensity at the detector after the flash is
recorded [Fig. 1(C)]. Under the condition that the flashinduced perturbation is completely relaxed to equilibrium
before the next flash is initiated, this experiment can be
repeated at another mirror position. After many steps, a set
of time-digitized transients for each mirror position is
obtained which can be re-sorted into a set of interferograms
for each time point. After Fourier transformation, complete
infrared spectra for each time point can be obtained
(Uhmann et al., 1991; Palmer et al., 1993; Rammelsberg
et al., 1997). Other time-resolved (not FT) techniques exist,
which have been recently reviewed by Slayton and Anfinrud
(1997).
Most of the infrared bands of proteins are relatively broad.

Resolution enhancement can be achieved by deconvolution
of the spectra (Surewicz and Mantsch, 1996). The basic idea
is that the spectrum S(n) is regarded as a convolution S'(n)
* L(n) of a delta function S'(n) = (n no) and a Lorentz
function L(n) = a/(p[a2 n2]) with a being the Lorentz halfwidth. A delta function corresponds to a cosine function in
the interferogram, S'(n) I'(n) = cos(2pnox), while a Lorentz function is described by an exponential in the
interferogram, L(n) `(x) = exp[2pajxj]. In the interferogram domain, the convolution product represents a simple
multiplication operation I(x) = I'(n)`(x) = cos(2pnox)exp
[2pajxj]. Resolution enhancement is reached by dividing
the interferogram I(x), obtained by inverse Fourier transformation of the spectrum, by the interferogram of the
Lorentz function. If the resulting interferogram is again
Fourier transformed, a spectrum with a higher resolution is
obtained. Using this procedure, the broad bands with a weak
fine structure can be resolved more clearly [Fig. 3(A)]. The
frequency of the band maxima in the deconvoluted spectra
should correspond to the frequency of the minima in the
second derivative of the undeconvoluted spectrum [Fig.
3(A)].
Nonlinear least-square curve fitting with given band
shapes is an alternative and/or additional method to
decompose broad structured bands. Different line shapes
are used depending on the kind of spectra and experiments.
J. Mol. Recognit. 2000;13:325351
328
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Figure 3. Analysis of infrared spectra. (A) Bottom: original amide I' band of
cytochrome P450cam from Fig. 2 and side-chain absorption spectrum calculated
according to the amino acid composition of cytochrome P450cam using the standard
spectra provided by Chirgadze et al. (1975); middle: side-chain corrected spectrum
corresponding to the difference of the original spectrum minus the side-chain
spectrum, deconvoluted spectrum of the side-chain corrected spectrum; top: second
derivative of the side-chain corrected spectrum. (B) Result of a nonlinear least-square
t of the deconvoluted amide I' band using Gaussians and assignment to secondary
structure elements. (C) Comparison of the line shapes of Gauss [E(n) =
2
2
Aexp[4ln2((n
no)/Dn1/2) ]], Lorentz [E(n) = A/[1 4((n no)/Dn1/2) ]] and Voigt
[E(ni) = (1/p) exp[(n ni)2/X22][X1/(X12 (n no)2)]dn, with X1 = 12Dn1/2(Lorentz); X2 =
1
1/2
Dn1/2(Gauss) and X1/X2 = shape factor; X1/X2 = 0 for Gauss; X1/X2 ? for
2(ln2)
Lorentz]. (D) Line shape of the logarithmic normal distribution function [E = (A/X)
exp[lnX(1 ln2lnX/ln2a)] with X = [(n no)(a2 1) Dn1/2a]/Dn1/2a and a = asymmetry
parameter; a < 1 asymmetry at the lower-energy side; a > 1 asymmetry at the
higher-energy side] for an asymmetry parameter of 1.4. [n = wavenumber; Dn1/2 = half
width; no = wavenumber of band maximum; E(n) = absorbance; A = amplitude].
Gauss and Lorentz line shapes are used for fitting the protein
amide bands [Fig. 3(B)]. For analysis of the stretch mode
spectra of the CO ironligand the Voigt function [convolution of a Lorentz and Gauss function; Fig. 3(C)] and the
logarithmic normal distribution function [Fig. 3(D)] have
also been used. The Lorentz line shape represents the
homogenous broadening of a spectral line and the halfwidth is a measure of the lifetime of the excited state. The
homogenous line width of the stretch mode of the CO ligand
in heme proteins lies in the range of 0.22 cm1 (Rector et
al., 1997). The Gaussian distribution results from the
interaction of the vibrating group with the environment
resulting in many microstructures for the vibrating group
(inhomogenous broadening effect). Because the width of
CO stretch modes in heme proteins is significantly larger
than 2 cm1 (720 cm1) one can conclude that inhomoCopyright # 2000 John Wiley & Sons, Ltd.
genous broadening is always present. The band shape must

be described by the Voigt function. A shape factor [Fig.
3(C)] allows a smooth change from a Lorentz to a Gauss
function when it is varied during the fitting. It is therefore a
measure of the extent of inhomogenous broadening. Serious
care has to be taken when fitting complex spectra with many
bands. The minimum number of bands resulting in a
reasonable fit with well-distributed positive and negative
wings in the residuals over the whole spectral region should
be considered [Fig. 3(B)]. Sometimes the absorption bands
are asymmetric in shape without a clear shoulder and the
result of fitting with two or more Gaussians is not
unequivocal and depends on the starting parameters. In
these cases, the bands can be fitted with the logarithmic
normal distribution function (Jung et al., 1996a). There is no
physical meaning behind this line shape but one can use it as
J. Mol. Recognit. 2000;13:325351
329
Table 1. Amide vibrations of the peptide group in proteins (Susi, 1972; Krimm and Badekar, 1986; Baello et al.,
1997)
Wavenumber (cm1)
In H2O
In D2O
Amide A
Amide I
32503300
16001700
16001700
Amide
Amide
Amide
Amide
Amide
1550
12301330
625767
640800
200
Band
II
III
IV
VI
VII
1450
Assignment
NH stretch, in resonance with amide II overtone
Mainly C=O stretching, slightly coupled with CN stretching, CCN
deformation, NH bending
NH bending coupled with CN stretching.
NH bending and CN stretching
OCN bending, coupled with other modes
Out-of-plane NH bending
Skeletal torsion
mathematical description for an ensemble of bands which

might be assigned to an ensemble of conformational states.
Various other methods are used to decompose the complex
infrared spectra (for example factor analysis, (Gillette et al.,
1983; Lee et al., 1990) which will not be further discussed
here.
A very powerful method for analyzing complete sets of
FTIR spectra for various perturbations like H/D exchange,
varying concentrations of a chemical agent, temperature
change or even time-resolved processes is the recently
developed two-dimensional FTIR spectroscopy (2D-FTIR)
which will certainly improve the analysis of complex
processes significantly (Nabet and Pezolet, 1997).
Before all these fitting and deconvolution procedures can
be applied to the experimental spectra, two corrections have
to be made. The first correction is necessary if the infrared
spectrometer is not purged carefully with dry air and the
water vapor absorption overlays with the sample spectrum
[Fig. 2(B)]. The water vapor spectrum can be interactively
subtracted using software. The second correction concerns
the baseline. For example, for studies of the dependence of
the iron ligand CO stretch modes on temperature in the
range 20297K, serious baseline shifts may be present if the
reference spectrum is not taken at the same temperature. A
baseline correction can be performed by fitting the spectrum
on the left side and the right side of the CO bands by a cubic
polynomial. Baseline problems are not so serious in
flashphotolysis or electrochemical experiments because
the spectra to be substracted are produced in the same
sample. Therefore, signals with a very weak absorption
(105) are still well detected.
INFRARED SIGNAL ASSIGNMENT

Proteins
Infrared spectroscopic studies on proteins have been
reviewed extensively (Surewitz and Mantsch, 1996; Arrondo and Goni, 1999). Besides the fingerprint vibrations of
functional groups of chemicals in general, which are
tabulated in various textbooks, there are specific vibrations
resulting from the peptide group (Table 1).
Amide I band. The best studied infrared band of proteins is
the amide I band appearing between 1600 and 1700cm1

with a maximum for most proteins at around 1654
1674 cm1 [Figs. 2(B) and 3(A,B)], which arises primarily
from the stretch vibration of the peptide C=O group.
Normal mode analysis reveals that the C=O stretching
couples slightly with CN stretching, CCN deformation and
NH bending. Unfortunately, the HOH bending motion of
water almost coincides with the amide I band [Fig. 2(B)]
and makes studies in protonic aqueous solution difficult
(Rahmelow and Hubner, 1997). This problem is overcome
by using D2O as solvent. The D2O substitution leads to a
shift of the amide I band (amide I') between 2 and 9 cm1 to
lower frequencies depending on the particular protein (Tu,
1986).
Because the C=O group is involved in different
secondary structure elements via hydrogen bonding to the
peptide NH group, the experimentally observed amide I
band envelopes a multitude of single bands with different
frequencies which can be resolved as described above [Fig.
3(B)]. The large number of infrared spectroscopic studies in
combination with crystal structure and NMR analyses on
various proteins revealed some variability of the frequency
of the assigned component amide I bands. However, one can
generally classify the components between 1649 and
1658 cm1 to a-helices, 16201635 cm1 to intramolecular
b-sheets, and 16651690 cm1 to turns. b-Sheets show a
weak high-frequency component at around 1672 cm1
which will, however, not significantly contribute to the
absorption in this region (Byler and Susi, 1986). However,
aggregated proteins show intermolecular antiparallel bsheets with infrared absorption around 16141624 and
1684 cm1 (Ismail et al., 1992; Panick et al., 1999). Some
exceptions from this general assignment have been
discussed in the recent review by Arrondo and Goni
(1999) and by Heimburg et al. (1999). Table 2 summarizes
the assignment of the amide I components to secondary
structure elements for cytochrome P450cam the only
P450 for which the amide I band spectrum has been
published so far.
Secondary structure composition can be estimated from
the relative area of the single bands assigned to the different
structures assuming that the extinction coefficient for the
peptide CO stretch vibration is the same in all hydrogen
bonding structures. This is of course an approximation.
Nevertheless, the agreement with the secondary structure
J. Mol. Recognit. 2000;13:325351
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Table 2. Component band frequencies of the amide I' band of cytochrome P450cam (Mouro et al., 1997)
(1R)-Camphor-bound
1
Position (cm )
1599.6
1611.1
1622.1
1631.8
1640.7
1648.8
1658.2
1666.1
1673.2
1689.3
Substrate-free
1
Population (%)
Position (cm )
Population (%)
Assignment
2
2
9
12
14
16
24
2
17
2
1555.5
1610.8
1621.8
1634.6
1648.2
1658.6
1672.5
1688.7
3
4
8
23
24
20
17
1
Arg side chain

Arg, tyr side chain
b-Sheet
b-Sheet
a-Helix, 310-helix
a-Helix
a-Helix
Turns
Turns
Turns
composition obtained from CD measurements of the

electronic transitions of the peptide chromophor and from
crystal structure analyses is reasonably good (Byler and
Susi, 1986). For camphor-bound P450cam, for example,
54% a-helix 310-helix, 21% b-sheets and 21% turns have
been estimated by FTIR (Mouro et al., 1997), which is in
good agreement with 53%, 19% and 16%, respectively,
determined from the crystal structure. Quantitation of
secondary structure compositions for various proteins have
also been performed from second derivative spectra (Dong
et al., 1990) and from factor analysis (Lee et al., 1990).
Amide II band. The amide II band arises from NH bending
coupled with CN stretching. The band is observed around
1550 cm1 in H2O and shifts to about 1450 cm1 [amide II',
Fig. 2(B)] in D2O (Susi, 1972). Because of the strong
sensitivity of the amide II band frequency upon deuteration
this band is used to monitor H/D exchange between the
protein core and the solvent during unfolding of the protein
structure induced by various perturbations as for example
temperature or pressure (Surewicz and Mantsch, 1996;
Heremans, 1997).
Amide III band. The amide III band is found in the range
between 1220 and 1330 cm1 and results from NH bending
and CN stretching. The band position is very sensitive to the
secondary structure. a-Helix is reflected in the region 1293
1328 cm1, b-sheet in 12251250 cm1 and unordered
structures in 12571288 cm1 (Griebenow and Klibanov,
1995). However, because of its low intensity the amide III
band observed with infrared spectroscopy is of lower
diagnostic value. Some progress in improving the significance of the structural information from this band has
recently been reported using vibrational circular dichroism
measurements (Baello et al., 1997).
Modes of amino acid side chains. Infrared absorption of
amino acid side chains also appears in the spectral region of
the amide I and II bands and makes the extraction of
structural properties from the amide bands more complicated. Depending on the amino acid composition of the
protein and on the pH, the contribution of the side chain
absorption can vary dramatically and may cause up to about
30% of the amide I band integral intensity. Standard spectra
for the side chain absorption have been published by
Chirgadze et al. (1975) for D2O solutions and by

Venyaminov and Kalnin (1990) for H2O solutions. If the
amino acid composition is known these spectra may be used
to correct the protein amide I band before deconvolution and
decomposition in the single bands to obtain the content of
the different secondary structure elements [Fig. 3(A)].
For estimation of the secondary structure composition of
proteins the side chain absorption may be regarded as
disturbing contribution. However, for the analysis of
recognition phenomena, side chain infrared bands are of
high diagnostic value, e.g. to detect salt links by the infrared
absorption of the COOH group in aspartic or glutamic side
chains. In the protonated state the CO stretch vibration of a
COOH group appears in the region around 1760 cm1 in
H2O and is shifted to 1750 cm1 in D2O (Dollinger et al.,
1986). This position is significantly lowered by hydrogen
bonding and signals at different frequencies between 1695
and 1740 cm1 have been observed in various proteins
(Lubben et al., 1999; Iliadis et al., 1994; Hellwig et al.,
1999). The deprotonated COO group shows the asymmetric vibration between 1540 and 1620 cm1 while the
symmetric vibration is observed between 1300 and
1420 cm1 (Chirgadze et al., 1975; Venyaminov and
Kalnin, 1990). The simultaneous appearance of bands
between 17501695 cm1 and 15401620 cm1 or 1300
1420 cm1 with opposite sign in difference spectra obtained
from perturbation-induced conformational changes is a
strong indication of a change in the protonation state (Iliadis
et al., 1994). In contrast, a shift of the signal or the
appearance of a derivative-shape difference signal in the
narrower spectral range between 1700 and 1740 cm1 is
caused by a change in the strength of the hydrogen bond
(Lubben et al., 1999; Contzen and Jung, 1999).
Histidine may also be involved in salt bridge formation
with aspartates. Three bands of histidine are sensitive to
protonation of the imidazole nitrogen in hemoglobins
(Table 3, Gregoriou et al., 1995). CH and CN
stretching modes of histidine imidazole have also been
assigned (Table 3).
Tyrosine CC ring vibration is observed at around
1515 cm1 in D2O and is a very good marker band because
of sharpness and almost isolated spectral location. This band
can be used to normalize infrared spectra of proteins
(Arrondo and Goni, 1999). In addition, hydrogen bonding
from the tyrosine to other groups leads to a shift of this band.
J. Mol. Recognit. 2000;13:325351
331
Table 3. Amino acid side chain infrared absorption signals

Wavenumber (cm1)
Amino acid
H2O
D2O
Aspartic acid
1713
1584
1706
1567
1648
Arginyl
1716
1574
1712
1596
1678
1622
1670
1610
1673
Tyrosyl
1633
1518
Histidine
1498
1602
12401280
1596
1586
1515
1615
1500
1603
Glutamic acid
Asparaginyl
Glutaminyl
Tryptophan
Cysteine
1635
1608
1087
1104
1089
1101
1090
1106
1611
1575
1410
1630
1530
1412
3135-3145
1197
1103
1108
1101
1096
1107
34863491
24002700
1857
Assignment
CO stretch in COOD, pH(D) <4
COO asymmetric
CO stretch in COOD, pH(D) < 4
COO, asymmetric
CO stretch
NH2 deformation
CO stretch
NH2 deformation
CN3H5 asymmetric stretch
CN3H5 symmetric stretch
Ring motion, pD < 9
pD < 9
Ring motion, pD > 10
pD > 10
CO stretching
Ring motion
Protonated
Protonated
Protonated
Neutral
Neutral
Neutral
CH stretch histidine imidazole
CN stretch in Nt(4-MeIm)
CN stretch in Np(5-MeIm)
CN stretch in Nt, Np
(MeImH(D))
CN stretch in (MeIm)
CNt (DL-His)
CNp (DL-His)
Indole N-H stretch
SH stretch
The SH sulfhydryl group of cystein absorbs between

2400 and 2700 cm1 in H2O and around 1857 cm1 in D2O
(Tu, 1986; Gregoriou et al., 1995; Guarrera et al., 1999).
SH groups which are hydrogen bonded show significantly
lower SH stretch modes down to 2290 cm1 (Sellmann et
al., 1991; Boorman et al., 1992). Table 3 summarizes the
infrared signals observed for amino acid side chains.
Cofactors
Proteins may have various cofactors such as flavins, iron
sulfur cluster, retinal or heme. On one hand, their infrared
absorption may complicate the interpretation of the infrared
spectra of proteins but on the other hand their specific
signals may be very helpful for the analysis of recognition
phenomena.
Retinal. Retinal is the cofactor in bacteriorhodopsin. The
signal of high diagnostic value is the Schiff base C=N
stretching mode located at about 1640 cm1 whose concrete
Reference
Venyaminov and Kalin (1990);
Chirgadze et al. (1975)
Venyaminov and Kalin (1990)
Roepe et al. (1987)

Venyaminov and Kalin (1990)
Gregoriou et al. (1995)
Puustinen et al. (1997)
Noguchi et al. (1999)
Maeda et al. (1992)

Tu (1986)
position depends on the protonation state of the nitrogen.

Other important signals are the CC stretch mode at about
1188 cm1 and the ethylenic C=C stretching at about
1527 cm1 of the carbons 13, 14, 15 next to the C=N group,
which are influenced by the isomerization state of the retinal
(Bousche et al., 1992).
Heme. Metal porphyrins show several vibrational modes in
a broad spectral region (Boucher and Katz, 1967; Alben,
1978; Spiro, 1983). Only the in-plane vibrations of the
symmetry Eu and the out-of-plane vibrations of the
symmetry A2u of the porphyrin ring are infrared active. In
most heme proteins the chromophore is protoporphyrin IX,
which has two vinyl groups. Infrared bands for the vinyl
group have been reported by Spiro (1983), the most
important one being the C=C stretch mode at about
1640 cm1. These vibrational modes are difficult to see in
the infrared spectra of heme proteins because of the overlap
with the amide bands of the protein. However, in difference
spectra obtained from photochemical or electrochemical
reduction or ligand photodissociation, small signals might
J. Mol. Recognit. 2000;13:325351
332
C. JUNG
Figure 4. Sketch of the binding mode of heme iron ligands and structural parameters: (A) in protein-free heme iron
complexes and assignment of the range of the ligand stretch mode frequencies (Collman, 1977; Alben, 1978; Yu, 1986;
Bof et al., 1997; Obayashi et al., 1997), and (B) in heme proteins indicating the parameters which inuence the stretch
mode frequency of heme-bound ligands (p and s indicate the iron and ligand orbitals assigned to the p- and s-orbital
system. The arrows point in the direction of the electron density donation in the respective orbital system. X = Y is the
distal iron ligand while `5. ligand' means the proximal iron ligand. and indicate the sign of the electrostatic
potential).
originate from porphyrin or its substituents (Schlereth and

Mantele, (1992); Miller and Chance, 1994).
Protoporphyrin IX has two propionic acid groups which
have characteristic infrared bands depending on the
protonation state of the COOH group, as already discussed
above for the COOH group in aspartate or glutamate. The
stretching mode of the CO group in the methyl propionate
ester has been observed at 1738 cm1 (in chloroform) and
between 1725 and 1740 cm1 for various other substituted
porphyrins (Alben, 1978). Two signals at 1735 and
1700 cm1 exist when the ester is split (Boucher and Katz,
1967). The CO stretch is coupled with the OH in-plane
bending in the COOH. Hydrogen bonding from the
propionic acid OH group or from a hydrogen donor to the
CO propionic group by basic amino groups in the protein
shifts the value to lower frequencies, 16971740 cm1
(Behr et al., 1998).
Ligands
Metal ligands. Infrared absorption of small heme iron
ligands like CO, O2, NO, CN, SCN, N3 have been
extensively studied for various heme proteins over several
decades, but other metal centers in proteins are also able to
bind such ligands. Before drawing conclusions about
proteinligand recognition in heme proteins one has to
remember the prefered mode of binding of these ligands in
protein-free metal complexes which follow the rules for
orbital hybridization. Binding of the ligand to the metal
changes the ligand stretch mode frequency [Fig. 4(A)].
Carbon monoxide. CO binds to ferrous heme and has a
linear geometry. The sp hybridization of the CO valence
orbitals would best describe the structure.

Dioxygen. O2 also binds only to heme with ferrous iron and
is in a bent configuration with a typical angle of
approximately 110130 because of the sp2 hydridization
(Godbout et al., 1999). The n(OO) stretch vibration of the
free ligand is not infrared-active because of a zero-dipole
moment. After binding to the heme iron the OO bond
becomes asymmetric and a nonzero dipole moment is
induced giving rise to a weak infrared absorption intensity.
O2 complexes have not been so extensively studied by
infrared spectroscopy because several other components of
the protein and solution absorb in the spectral region of the
OO stretch mode and the OO band intensity is low.
Resonance Raman spectroscopy is an alternative method
(Bajdor et al., 1984: Macdonald et al., 1999).
Nitric oxide. IronNO complexes usually show a bent
geometry if the iron is in the ferrous state [rule given by
Feltham and Enemark (1981): {M NO}n, M = metal,
n = number of the electrons in the metal d-orbitals and the
p* orbital of the NO ligand; for n 7 MNO is bent]. In
this case NO can accept an electron from iron(II) by forming
the resonance structure (Fe3NO) with NO being
isoelectronic with O2. The n(NO) stretch vibration of
the bound NO is therefore expected to be in a lower
frequency range down to 1550 cm1 of the free NO. In
contrast, ferric NO complexes prefer a linear ironligand
orientation (case n 6) by donating an electron to the iron
dp-orbitals and forming NO which is isoelectronic with
CO. In this case n(NO) should be in the range of 2150
2400 cm1 of free NO. In real complexes however the
stretch frequency lies between the frequency of free NO and
either NO or NO.
J. Mol. Recognit. 2000;13:325351
333
Cyanide. CN can bind to either ferric or ferrous heme

(Yoshikawa et al., 1985; Yu, 1986; Boffi et al., 1997). The
CN stretch vibration frequency of the free CN and HCN
are observed at about 2078 and 2092 cm1, respectively.
When bound to ferric complexes it shifts up because of s- as
well as p-donation to the iron, but shifts down at binding to
ferrous heme because of increased iron-p-backdonation.
Undisturbed cyanide complexes have a linear FeCN
geometry.
molecules to the protein is mediated by hydrogen bonding

which significantly influences the OH stretch vibration of
H2O. The antisymmetric OH stretch mode of water
appears at about 3550 cm1 [Fig. 2(B)] but may shift up or
down depending on the microenvironment in the protein.
Specific water OH stretch changes can only be observed
in thin films and in difference spectra induced, for example,
by illumination as studied in bacteriorhodopsin (Maeda et
al., 1993).
Azide. Free azide is linear with symmetric NN bonds.

When bound the metal complex N3 remains in the linear
fashion but the NN bonds become asymmetric. The linear
N3 ligand binds in a bent mode to the metal. For iron heme
complexes it binds to the ferric form.
Phosphate. ATPases, kinases, and ion pumps bind phosphate and nucleotides. Phosphate buffer is commonly used
for protein solution. Knowledge of the infrared absorption
signals of phosphate is therefore important. The asymmetric
and symmetric stretch vibrations of PO2 in H2PO4 are
located at 1158 and 1068 cm1, respectively. The symmetric stretch and the degenerate stretch of PO22 in
H2PO42 appear at 992 and 1068 cm1, respectively. These
signals are influenced when phosphate binds to proteins
(Cepus et al., 1998a,b).
Environmental effects. The concrete value of the ligand

stretch mode frequency is determined by various parameters
[Fig. 4(B)]. The most important one is the balance between
electron density distribution within the s- and p-systems of
the metalligand unit. This has been extensively discussed
in the literature for the CO and O2 complexes as the synergic
effect of ligand-s-donation and iron-p-backdonation (Yu,
1986). This balance is sensitive to various other parameters
such as the nature of the proximal (5th) ligand, the angle
between the ironligand and the intra-ligand bonds,
hydrogen bonding, electrostatic interaction between the
ligand and amino acids, substrates and water on the distal
side of the porphyrin plane. Also the porphyrin substituents
influence the stretch frequency (cis-effect) due to their
electronic effect on the porphyrin-p-system (Alben and
Caughey, 1968; Alben, 1978).
Studies on pyridinehemeCO complexes showed that
the n(CO) decreases when the basicity of the trans-pyridine
ligand increases (trans effect) (Alben and Caughey, 1968).
The same trend has been observed in model complexes
when the proximal imidazole or mercaptane are deprotonated (Mincey and Traylor, 1979; Chang and Dolphin,
1976). The reason is that the stronger charge donation into
the antibonding orbitals of the CO or O2 ligands leads to an
increase of the ligand net charge and a decrease of the pbond order. Yoshimura (1983) has shown for NO complexes
of ferrous protoporphyrin IX with nitrogenous bases as
proximal ligand that, with increasing the polarity of the
solvent, the stretch mode frequencies decrease. These
effects of the proximal ligand and of the polarity of the
environment observed in porphyrin complexes represent
already the range of effects which turn out to be the most
important ones for the stretch mode frequencies of the
ligands bound in heme proteins, as discussed below.
Other ligands
Bound water. Infrared studies of proteins in protonic
aqueous solution are problematic because of the HOH
bending absorption at about 1650 cm1 which overlaps with
the amide I band as discussed above. However, specific
water molecules play an important role for the structure of
proteins and have functional significance for proton
translocation (Yamazaki et al., 1995). One may understand
these water molecules as ligand. Binding of water
Recognition phenomena
Recognition phenomena in heme proteins with
histidine as proximal iron ligand
Hemoglobins and myoglobins. CO binds to protein-free
heme with an affinity which is approximately 20000 times
higher than that for O2. However, in proteins such as
hemoglobin and myoglobin this ratio is dramatically
lowered to about 25200. How can hemoglobins and
myoglobins as important oxygen carrier and storage
proteins discriminate between dioxygen and other small
ligands which act as poison such as CO, CN and NO?
CO stretch vibration: FeCO bending or electrostatic
polarization? For a long time it was thought that the protein
destabilizes the CO bond to the heme iron by steric
hindrance. This had been concluded from the FeCO
geometry, seen in the crystal structure of heme proteins,
which deviates from the expected linear orientation.
Infrared spectroscopic studies seemed to support this idea
at first view. In myoglobin, essentially three CO stretch
mode bands [Ao (1965 cm1), A1,2 (19451954 cm1),
and A3 (1932 cm1)] are observed with different relative
integral intensities of the bands (Ansari et al., 1987). In
hemoglobin from various sources, a major CO stretch band
between 1948 and 1951 cm1 and minor bands in the region
around 1930, 1943 and 1970 cm1 are observed (Potter et
al., 1990). Frauenfelder and coworkers have explained the
different infrared bands with conformational substates (A
states) of the protein (Frauenfelder, 1997). Ormos et al.
(1988) and Moore et al. (1987) assigned them to different
FeCO angles by measuring the linear dichroism
following photoselective flashphotolysis of the CO ligand
using infrared spectroscopy at low temperatures or timeresolved. However, recent infrared dichroism and timeresolved infrared polarization spectroscopic studies on COmyoglobin revealed an angle of 7 from the normal heme
without differences for the subconformer A states (Ivanov et
al., 1994; Sage and Jee, 1997; Lim et al., 1995a). The recent
crystal structure at near-atomic resolution (Vojtechovsky et
J. Mol. Recognit. 2000;13:325351
334
C. JUNG
Figure 5. Sketch of the heme pocket in heme proteins indicating the interaction of the
CO ligand with distal side groups and the assignment of the CO stretch mode. (A)
Myoglobin (according to Vojtechovsky et al., 1999); (B) horseradish peroxidase (the
propionic group HOOCprop. is involved in a hydrogen bond network with Gln176,
Ser73, Ser35 and Arg31, according to Gajhede et al., 1997); and (C) cytochrome c
oxidase; the lower frequency belongs to the iron-bound CO and the higher frequency
to the copper-bound CO (according to Mitchel et al., 1996).
al., 1999) is in agreement with this observation. This crystal

structure and infrared studies where amino acids have been
mutated on the heme proximal side (L89I and H97F)
demonstrate that the different A substates are predominantly
caused by the distal side (Abadan et al., 1995) and only the
population of A3 is affected by the influence of the proximal
side, as shown for the mutant H93G (Decatur et al., 1996).
Various mutations on the distal side of different myoglobins
demonstrated that the frequency range for the three bands is
maintained and only the population of the subconformer
states is affected (Braunstein et al., 1993; Cameron et al.,
1993; Li et al., 1994; Anderton et al., 1997; Uchida et al.,
1997a,b; Hildebrand et al., 1998). For some of these mutants
the crystal structure has been resolved, revealing almost the
same FeCO geometry.
At present most of the data indicate that the distal
histidine (His64) in its three orientations of the imidazole
modulates the dipole moment of the bound CO ligand by
hydrogen bonding and/or electrostatic interactions without
having a significant effect on the FeCO angle (Ray et
al., 1994; Springer et al., 1994; Rector et al., 1997). Recent
theoretical studies support this conclusion (Kushkuley and
Stavrov, 1996, 1997a,b). Ao has been assigned to the
protonated histidine in its swung-out conformation [Vojtechovsky et al., 1999; Fig. 5(A)]. A1 is identified as a
histidine conformation with the lone pair of the imidazole
nitrogen atom pointing to the CO ligand with a nitrogen
causing predominantly polar
oxygen distance of 3.2 A
interaction. A3 should correspond to the histidine conformation with the NH pointing in the direction of the CO oxygen
and forming a hydrogen bond.

atom at a distance of 2.7 A
Alternative assignments of the Ao, A1 and A3 states to the
histidine conformations have been performed by different
authors, as discussed by Vojtechovsky et al. (1999). The
deviation of the FeCO geometry from the linearity
(tilting and small bending), as generally observed in heme
proteins, has been explained using ab initio calculations by
the effect of the proximal ligand (Jewsbury et al., 1994).
CO a good model for O2? Studies of CO complexes of
myoglobins and hemoglobins are motivated by regarding
CO as a model for O2 because both ligands bind to the
ferrous heme at the same coordination position. However in
contrast to CO, O2 generally binds in a bent orientation [Fig.
4(A)]. Potter et al. (1987) have found two OO stretch
mode signals at 1150 and 1120 cm1 for bovine oxymyoglobin with a minor component at about 11031107 cm1.
Miller and Chance (1994) have recently shown by
photolysis difference infrared spectroscopy that the OO
stretch band at around 1103 cm1 overlaps with CH
bending mode of the imidazole in histidine. The two major
substates at 1150 and 11251135 cm1 have been recently
assigned by high-resolution crystallography to subconformations of the distal histidine in myoglobin (Vojtechovsky
et al., 1999). The 1135 cm1 signal should belong to the
distal histidine conformation which may form a hydrogen
bond to the terminal oxygen atom of the O2 ligand (A3 in CO
complex) while the other OO mode is assigned to the
histidine conformation which induces polar contact [A1 in
CO complex; Fig. 5(A)]. An OO stretch mode, corresponding to the A0 conformation without H-bond and polar
J. Mol. Recognit. 2000;13:325351
contact, does not seem to exist.

There is consensus between the various researchers that
steric constraints on the bound CO ligand by the protein are
not relevant for discrimination from O2 binding. The
preferred explanation is that dioxygen can be better
stabilized by hydrogen bonding or enhanced electrostatic
interactions because of the larger negative net charge at the
terminal oxygen atom (Olson and Phillips, 1997; Borman,
1999).
H bonding and/or electrostatic interactions for NO, N3
and CN ? NO can bind to the ferric as well as ferrous state.
Crystal structure analyses of NO-ferrous form of hemoglobins and myoglobins revealed a bent FeNO geometry
with an angle between 112 and 147 (Harutyunyan et al.,
1996; Brucker et al., 1998) which matches the angle of 131
149 reported for NO ferrous tetraphenylporphyrin compounds (Scheidt et al., 1977). Corresponding to this
geometry and in agreement with the rule by Feltham and
Enemark (1981) the NO stretch vibration observed lies
between that of NO and NO (Fig. 4; 16071615 cm1;
Miller et al., 1997). However for ferric myoglobin, the NO
stretch vibration has been observed at 1927 cm1 in the
difference infrared spectrum produced by photodissociation
of NO (Miller et al., 1997). This value is in between the
frequency of NO and NO. This frequency decreases to
1904 cm1 when His64 is replaced by leucine. In contrast,
the stretch mode of the CO ligand in the ferrous myoglobin,
which is isoelectronic to the ferric NO complex, shifts the
population from the lower-frequency mode A1 to the higherfrequency mode A0 upon mutation. This example shows that
hydrogen bonding and electrostatic interactions are very
important. Ferric NO complexes may be described by a
formal charge distribution of FedNOd in contrast to
FedCOd. An inverse response of the ferric NO and the
ferrous CO complexes on His64 mutation is therefore
expected.
Azide myoglobin and hemoglobin exist in an equilibrium
of iron(III) low-spin and high-spin states, which is reflected
in the N3-vibration mode. Studies by Alben and Fager
(1972) and McCoy and Caughey (1970) assigned the bands
at about 2023 and 2046 cm1 to the low-spin and the highspin states, respectively. The N3 stretch frequency shows a
small shift of the low-spin state band to lower frequencies
when a positive charge is introduced on the protein surface
near the heme pocket (Val67Arg) or to higher frequencies
for a mutation to a negative charge (Lys45Glu); Bogumil et
al., 1994). This indicates that electrostatic interactions may
stabilize or destabilize the azide ligand binding, respectively. Mutations of the distal His64 induce stronger
changes of the low-spin state corresponding stretch mode
(down-shift by 58 cm1 for the Thr and the Ile mutants and
a significant increase of the band width). This suggests that a
hydrogen bond between the distal histidine and the azide
ligand in the low-spin state should exist, which is affected
by mutations. In contrast to N3 complexes, however, the
CO stretch mode is more affected by mutations, as recently
observed for a quadruple mutant (Thr39Ile/Lys45Asp/
Phe46Leu/Ile107Phe) of horse heart myoglobin (Hildebrand
et al., 1998), indicating that CO is a better sensor for the
polarity on the distal side.
Boffi et al. (1997) have studied the Fe(II) and Fe(III)
cyanide complexes of homodimeric Scapharca inaequivalCopyright # 2000 John Wiley & Sons, Ltd.
335
vis hemoglobin. The CN stretch mode for the Fe(III)

complex is observed at 2132 cm1 and for the Fe(II)
complex at 2058 cm1. Uchida et al. (1997a) found for
various mutants at position 29 of Fe(III)-myoglobin the CN
stretch mode between 2125 and 2135 cm1. There is little
variation in the CN stretch frequency in the Fe(III)
complexes of heme proteins (Reddy et al., 1996) because
the CN binding to Fe(III) is dominated by the s-donation
and little contribution of p-backdonation exists (Kushkuley
and Stavrov, 1997b). In contrast, the additional electron in
the Fe(II) complexes is distributed over the s and p* orbitals
of CN resulting in a larger negative net charge on the
nitrogen atom of CN, which favors electrostatic interactions, and in the decrease of n(CN).
Summarizing, the ligand stretch mode for the different
distal iron ligands is predominantly determined by their
general binding mode to the metal porphyrins but fine-tuned
by hydrogen bonding and electrostatic interaction to the
distal side histidine in the different orientations. Among the
different ligands CO seems to be most sensitive to the
polarity on the distal side while hydrogen bonding may be
more important for O2. The other ligands are in between or
less affected. However, it is difficult to estimate the concrete
relative contribution of these both interactions for stabilizing the bound ligand.
Peroxidases. Peroxidases form a large family of heme
proteins which catalyze the oxidation of various substrates
using hydrogen peroxide as oxidizing agent whose OO
bond is heterolytically split (Dunford and Stillman, 1976).
Two recognition phenomena occurthe binding of hydrogen peroxide to the heme iron, and the binding of the
substrate. There is no high substrate specificity. Chemical
and crystallographic data indicate that the access of the
heme pocket for small substrates is restricted. H2O2 binding
to the heme iron is the more important recognition
phenomenom. The split of the OO bond in hydrogen
peroxide is an acidbase catalyzed process where histidine
on the distal heme side serves as the proton acceptor. The
H2O2/protein recognition cannot be studied with conventional methods because of the short life time of the Fe
H2O2 complex (Miller et al., 1994). Therefore, iron ligands
like CO, O2, NO and CN have been used as spectroscopic
probes to study the capability of the distal side to donate or
accept a proton.
Among the peroxidases, horseradish peroxidase is one of
the best studied proteins. Infrared studies on different
isozymes indicated two CO stretch modes, around
1905 cm1 at low pH and around 1932 cm1 at high pH
(Barlow et al., 1976). H2O/D2O exchange (Smith et al.,
1983) and pH dependence infrared studies clearly indicated
that the low-frequency mode corresponds to a hydrogen
bond between CO and an amino acid side chain which has
been shown to be a histidine [Gajhede et al., 1997; Fig.
5(B)]. Both conformers are interconvertible either by pH or
by binding of the substrate (benzhydroxamate; Uno et al.,
1987). Distal and proximal sides are strongly coupled by a
hydrogen bond network involving various amino acid side
chains, a water molecule and the heme propionate [Gajhede
et al., 1997; Fig. 5(B)]. This may contribute to the strongly
lowered CO stretch mode frequency at low pH. The stretch
mode of the CN ligand has been taken as a better probe
J. Mol. Recognit. 2000;13:325351
336
C. JUNG
Figure 6. Correlation of the iron-bound CO and the FeCO

stretch mode frequencies for various heme proteins and
porphyrinironCO complexes. Data are taken from Table 1 in
Ray et al. (1994) for the weak proximal ligand (benzene,
tetrahydrofuran) and for various hemoglobins and myoglobins
for the imidazole ligand and from Legrand et al. (1995) for the
thiolate ligand (cytochrome P450camCO in the presence of
various substrates).
than the CO ligand because it approches the iron as HCN

and donates the proton to the distal histidine when
coordinating to iron. This mimics the situation when
hydrogen peroxide binds. Bound CN exists in two
conformations (almost linear and tilted, respectively)
suggested from resonance Raman studies. In both conformers CN is suggested to be hydrogen bonded to the
distal histidine and/or to other distal groups (Tanaka et al.,
1997). The CN stretch mode frequency is therefore very
sensitive when structural changes are induced by mutations
around the histidine.
FTIR studies on yeast cytochrome c peroxidase, another
member of the peroxidase family, show that similar to
horseradish peroxidase two conformers in the CO complex
exist which interconvert by changing the pH. However, the
CO stretch frequency of the conformer at low pH,
corresponding to the 1905 cm1, conformer in horseradish
peroxidase, is up-shifted to 1922 cm1, indicating a much
weaker hydrogen bond to the distal histidine (His52;
Smulevich et al., 1986).
Oxidases. Oxidases catalyze the reduction of dioxygen to
water (Gennis, 1998). The active site consists of a heme
) to the heme
with a copper center very close (4.55.2 A
iron (binuclear reaction center; Michel et al., 1998). The
interesting recognition phenomena are the binding of
dioxygen in the binuclear reaction center and the transfer
of electrons and protons to O2 (Yamazaki et al., 1999). The
binuclear reaction center has been extensively characterized
also by FTIR using CO, NO, CN and N3 as spectroscopic
probes (Tsubaki et al., 1996, 1997; Zhao et al., 1994).
Depending on the redox state of the different redox centers
within the bovine heart cytochrome c oxidase, the stretch
frequency of the CO ligand is observed between 1959 and
1965 cm1 (Yoshikawa et al., 1977; Yoshikawa and
Caughey, 1982). The most striking finding is the very small
width of the CO stretch band <4 cm1 (compared to
hemoglobin >8 cm1), which has been explained with a

very stable and restricted CO binding arrangement in an
apolar environment (Fiamingo et al., 1982). Two CO
conformers are observed in the bacterial aa3-type cytochrome c oxidase from Rhodobacter sphaeroides with
resonance Raman as well as infrared studies (Wang et al.,
1995). The higher frequency n(CO) mode at 1966 cm1
corresponds to the n(FeCO) stretching mode at 519 cm1,
named the a-form, while the lower-frequency n(CO) mode
at 1955 cm1 is assigned to the n(FeCO) stretching
mode at 493 cm1 (b-form). Surprisingly, the a-form of
cytochrome c oxidase from different sources shows unusual
properties regarding the relation between n(CO) and n(Fe
CO). For heme carbonyl complexes with histidine as
proximal ligand there is an inverse linear relation between
n(FeCO) and n(CO) (Fig. 6; Li and Spiro, 1988; Ray et
al., 1994; Anderton et al., 1997). Cytochrome c oxidase lies
off from this line and is shifted to higher CO stretch
frequencies, normally observed only for weak proximal
ligands or ligand-free carbonyl porphyrin complexes,
although histidine as proximal ligand has been identified
in this protein.
The proximity of the copper center can be probed when the
CO ligand is photodissociated from the heme iron [Fig. 5(C)].
The dissociated CO is trapped on the copper and shows two
CO stretch modes for the copper-bound CO at 2037
2039 cm1 (corresponding to the b-form) and at 2061
2064 cm1 (corresponding to the a-form) in case of bovine
heart cytochrome c oxidase and of the aa3-type cytochrome c
oxidase from Rhodobacter sphaeroides (Fiamingo et al.,
1982; Wang et al., 1995). The a- and b-form differ in the
polarity around the binuclear center (Mitchell et al., 1996).
Iwase et al. (1999) have recently reported time-resolved
FTIR studies of the CO photodissociation of bovine
cytochrome c oxidase. The light minus dark difference
spectrum indicated the CO stretch of CuBCO at
2063 cm1. A negative band at 1737 cm1, resulting from
the CO stretch in protonated COOH, and a positive band
between 1550 and 1600 cm1, corresponding to the asymmetric stretch mode of COO, suggests that deprotonation of
a carboxylate (presumably Glu242) is accompanied by the
photoinduced CO transfer from the heme iron to the copper.
Puustinen et al. (1997) have recently shown in a very
elegant way how FTIR can help to uncover recognition
phenomena. The light minus dark difference infrared
spectrum at 80K has been measured for the carbon
monoxide complex of cytochrome bo3 oxidase from
Escherichia coli and its Glu286Asp and Glu286Cys
mutants. Signals in different spectral regions, corresponding
to different structural sites in the protein, are simultaneously
monitored: (i) CO stretch mode at 1960 and at 2065 cm1,
for CO bound at the iron and the copper, respectively [Fig.
7(A)]; (ii) a derivative-shape spectral change around 1724
1731 cm1 assigned to a changed hydrogen bonded
carboxylic acid C=O [inset in Fig. 7(A)]; (iii) a spectral
shift at 31453135 cm1 assigned to weakening of a
histidine imidazole CH stretching; and (iv) a derivative
spectral change at 3020 and 3080 cm1 resulting from the
CH stretch of the heme vinyl group or methine bridge
[Fig. 7(B)]. Signals (ii) are sensitive against mutations at
position 286 demonstrating a hydrogen-bonded connectivity
between Glu286 and a histidine copper ligand which may
J. Mol. Recognit. 2000;13:325351
337
Figure 7. FTIR `light minus dark' difference spectrum for the CO complex of
wild type cytochrome bo3 at 80 K (reprinted with permission from Puustinen et
al., 1997, copyright (1997) American Chemical Society). (A) spectral region of
the CO metal ligand stretch mode. [The CO stretch mode in the iron-bound
form and copper-bound form are seen as a negative band at 1960 cm1 and a
positive band at 2065 cm1, respectively. Simultaneously, a derivative-shape
spectral change is observed at 1724 cm1 (negative signal) and at 1731 cm1
(positive signal; inset) assigned to a weaker hydrogen bonded carboxylic acid
C=O in the complex when CO is bound to the copper center. This spectral
feature shifts to 1756 and 1761 cm1, respectively, when Glu286 is substituted
by Asp and is completely lost for the Glu286Cys mutant.] (B) A spectral shift is
observed at 31453135 cm1 upon CO binding to copper CuB, which can be
assigned to weakening of a histidine imidazole CH stretching. In addition, a
derivative spectral change is seen at 3020 and 3080 cm1 resulting from the
CH stretch of the heme vinyl group or methine bridge.
play a role in the proton translocation and therefore for the

dioxygen reduction to water. Analogous studies have been
performed on bd-type ubiquinol oxidase from Escherichia
coli using CN as probe molecule. These studies show that
CN is released from the complex upon photoreduction and

that a change of the hydrogen bonding of a cysteine SH,
monitored at the shift of the SH stretch mode, occurs
(Yamazaki et al., 1999).
J. Mol. Recognit. 2000;13:325351
338
C. JUNG
Table 4. CO stretch mode frequencies for the CO complex of cytochromes P450 at room temperature (Gauss curve
t data in case of overlapping bands)
n(CO) (Dn1/2, population) (cm1)
Protein
P-450lm2 (CYP2B4)
P-450lm4
P-450rlm2 (CYP2B1)
P-450rlm2 (CYP2B1)
P-450rlm4
P-450rlm4
P-450scc (CYP11A1)
Substrate-free, Adx
Substrate-free, Adx
Cholesterol, Adx
Cholesterol, Adx
25-OH-Cholesterol, Adx
25-OH-Cholesterol, Adx
22(R)-OH-Cholesterol, Adx
22(R)-OH-Cholesterol, Adx
22(S)-OH-Cholesterol, Adx
20,22-(OH)2-Cholesterol, Adx
22-Ketocholesterol, Adx
22-Ketocholesterol, Adx
P-45011b (CYP11B1)
Deoxycorticosterone, Adx
Deoxycorticosterone, Adx
P-45015b (CYP106A2)
Substrate-free
deoxycorticosterone
P-450lin (CYP111)
Substrate-free
H2O
1949 (1516)
1954 (15), 1961(s)
1948 (25)
1948
1952
1954 (30)
D2O
Reference
1949 (14)
1954 (1314), 1961
1948 (20)
1952 (21)
Bohm et al. (1976)

Bohm et al. (1979)
Bohm et al. (1979)
OKeefe et al. (1978)
Bohm et al. (1979)
1952.5 (12.6)
1951.5 (12.4)
1953.7 (12.9)
1951.9 (12.5)
1954.7 (9.9)
1954.6 (10.4)
1951.8 (12.6)
1934.5 (11.6)
1951.7 (12.6)
1933.4 (12.6)
1946.8 (10.4)
1946.2 (10.7)
1949.5 (17.0)
1946.2 (17.7)
1937.2 (13.6)
1950.6 (13.0)
1949.0 (13.2)
Tsubaki
Tsubaki
Tsubaki
Tsubaki
Tsubaki
Tsubaki
Tsubaki
et
et
et
et
et
et
et
al.
al.
al.
al.
al.
al.
al.
(1992)
(1992)
(1992)
(1992)
(1992)
(1992)
(1992)
1937.3 (8.8)
1937.2 (9.1)
Tsubaki et al. (1992)

1937.5 (13.4, 0.156)

1946.3 (12.5, 0.581)
1957.4 (14,6, 0.231)
1968.6 (6.9, 0.023)
1984.0 (7.4, 0.009)
1936.0 (17.7, 0.568)
1947.2 (9.4, 0.068)
1960.1 (18.0, 0.289)
1976.6 (11.1, 0.075)
Simgen et al. (2000)

Tsubaki
Tsubaki
Tsubaki
Tsubaki
Tsubaki
Tsubaki
Tsubaki
et al.
et al.
et al.
et al.
et al.
et al.
et al.
(1992)
(1992)
(1992)
(1992)
(1992)
(1992)
(1992)
Simgen et al. (2000)
1944.3 (9.5)
1955.8 (6.5)
1965.9 (22.9)
1953.0 (10.0)
Jung and Marlow (1987)
(1R)-Camphor
1942 (1921)
1963 (1112)
1940 (13)
1939.8 (12.0, 0.52)
1949.4 (10.4, 0.07)
1955.3 (19.2, 0.41)
1939.8 (12.7)
1938.4 (18.6,
1953.1 (11.1,
1962.6 (14.3,
1940.2 (12.2,
(1S)-Camphor
1940.7 (9.5)
1941.4 (10.2, 1.00)
(1R)-Camphor quinone
(1S)-Camphor quinone
1940.5 (10.6)
1939.6 (9.1)
Linalool-bound
P-450cam (CYP101)
Substrate-free
(1R)-Camphor
Substrate-free, type 1
Jung and Marlow (1987)
0.44)
0.05)
0.51)
1.00)
Jung et al. (1996b); Legrand et al.

(1995)
(1995); Schulze et al. (1996)
(1995); Schulze et al. (1996)
Jung et al. (1996b)
Jung et al. (1996b)
J. Mol. Recognit. 2000;13:325351
339
Table 4. continued.
n(CO) (Dn1/2, population) (cm1)
Protein
Adamantanone
Adamantane
1-Azidoadamantane
1-Chloroadamantane
1-Bromoadamantane
1-Iodoadamantane
1-Bromodimethyl-adamantan
1-Chlorodimethyl-adamantane
Fenchone
Endo-borneol allyl ether

Camphane
Norcamphor
Norbornane
3-Endo-norborneol
Endo-borneol propyl ether
Tetramethylcyclohexanone
3-Bromocamphor
P-450cam mutant D251N,
1R-camphor bound
P-450cam Pdx
P-420
P-420lm2
P-420lm4
P-420rlm2
P-420rlm4
P-420cam
H2O
1941.5 (9.0)
1942.3 (9.1)
1955.0 (11.7, 1.00)
1928.6 (41.4, 0.162)
1939.9 (16.3, 0.241)
1955.0 (14.1, 0.597)
1945.5 (13.2)
1943.8 (8.3, 0.88)
1952.6 (10.0, 0.12)
1943.1 (8.4, 0.60)
1950.8 (13.1, 0.40)
1943.2 (9.9, 0.16)
1954.6 (11.6, 0.84)
1941.6 (11.1, 0.20)
1955.2 (10.9, 0.80)
1943.4 (12.2, 0.14)
1955.3 (10.9, 0.86)
1944.5 (13.8)
1944.7 (11.1)
1944.0 (10.5, 0.74)
1952.2 (12.4, 0.26)
1941.5 (15.7, 0.60)
1955.3 (10.8, 0.27)
1961.9 (21.0, 0.13)
1940.9 (10.5, 0.119)
1953.7 (14.0, 0.881)
1946.1 (10.3)
D2O
Reference
Jung et
Jung et
Jung et
Jung et
al.
al.
al.
al.
(1996b)
(1992b)
(1996b)
(1992b)
Jung et al. (1996b)

Jung et al. (1996b)
Jung et al. (1996b)
Jung et al. (1996b)
Jung et al. (1996b)
Jung et al. (1996b)
1944.1 (10.8)

(1995)
Jung et al. (1992b)
Jung et al. (1996b)
1942.9 (19.4, 0.29);

1951.9 (10.8, 0.71)

(1995)
Jung et al. (1992b)
1946.4 (9.8)
1947.0 (10.1)
1953.0 (10.0)
1951.5 (9.0)
1944.7 (8.0, 0.20)
1954.7 (12.0, 0.70)
1965.0 (12.6, 0.10)
1933.3 (10.9)
1934.2 (9.7)
1933.7 (10.6)
Jung et al.
(1995)
Jung et al.
Jung et al.
Jung et al.
Jung et al.
(1996b); Legrand et al.

(1992b)
(1996b)
(1996b)
(1996b)
Jung et al. (1996b)

Jung et al. (1992b)
1938.2 (10.4)
Contzen and Jung (1998)
1932
Unno et al. (1997)
1966 (20)
1972
1966
1970
1964
Bohm et al. (1976)

Bohm et al. (1979)
Bohm et al. (1979)
Bohm et al. (1979)
Mouro et al. (1997)
Adx = adrenodoxin; Pdx = putidaredoxin.
Recognition phenomena in heme proteins with

cysteinate as proximal iron ligand
Three heme proteins have a deprotonated cysteine as the
proximal iron ligand: cytochromes P450, NO synthases and
chloroperoxidase. Although the heme complex coordination
is very similar, the secondary and tertiary structures are

completely different (Hasemann et al., 1995; Crane et al.,
1999; Fischmann et al., 1999; Li et al., 1999; Sundaramoorthy et al., 1993). In contrast to myoglobins and
hemoglobins, for which the recognition of the iron ligands
themselves is the point of interest, in the heme thiolate heme
J. Mol. Recognit. 2000;13:325351
340
C. JUNG
Figure 8. CO stretch mode infrared spectra of various substrate

complexes of cytochrome P450cam. (1R)-camphor (1); (1S)camphor (2); (1S)-camphor quinone (3); (1R)-camphor quinone
(4); adamantanone (5); 1-azidoadamantane (6); 1-chloroadamantane (7); fenchone (8); 1-bromoadamantane (9); endo-borneol
allyl ether (10); camphane (11); 1-iodoadamantane (12); norcamphor (13); norbornane (14); adamantane (15); 1-bromodimethyladamantane (16); 3-endo-norborneol (17); 1-chlorodimethyladamantane (18); endo-borneol propyl ether (19); tetramethylcyclohexanone (20) (Jung et al., 1996b).
proteins the substrate binding and recognition is an

additional important phenomenon.
Cytochromes P450. First infrared spectroscopic studies
were already carried out on a microsomal P450 in 1976
(Bohm et al., 1976) and on the bacterial P450cam in 1978
(OKeefe et al., 1978). Later, many studies have been done
on P450cam as function of substrate binding, solvent,
temperature and pressure. A summary of the CO stretch
modes, observed so far, is given in Table 4.
P450cam CO ligand stretch mode and substrate binding.
In the absence of any substrate a broad, structured band is
observed which results from the overlap of various
subbands (Jung and Marlow, 1987; Jung et al., 1992b,
1996a). P450cam has no amino acid side chain on the distal
side which could specifically interact with the CO ligand
and therefore cause the sub-bands as is the case in
myoglobin with the distal histidine. However, substrate
binding has a remarkable effect on the CO stretch mode
(Jung et al., 1992b, 1996b; Fig.8). The number of stretch
mode bands is restricted compared to the substrate-free
protein and the band frequencies strongly differ. Inspection
of the three-dimensional crystal structure of P450cam-CO
in the (1R)-camphor complex (Raag and Poulos, 1989)
reveals that the substrate is in van der Waals contact with the
CO ligand. Similar to the heme proteins with imidazole as
proximal ligand, the CO ligand is tilted (9) and slightly
bent 166 (Fig. 9).
In the first study on various substrate-P450cam complexes, Jung et al. (1992b) suggested that water molecules
in the heme pocket may interfere with the CO ligand and
might, therefore, be responsible for the different Fe-CO
conformers. The different subconformers were originally
Figure 9. Active site of (1R)-camphor-bound cytochrome

P450cam-CO. The coordinates are taken from the PDB entry
code 3cpp (Raag and Poulos, 1989). Sketch of camphor analogs.
assigned to different FeCO angles in analogy to data of

Ormos et al. (1988) for myoglobin. From a study of a large
number of substrate complexes, however, it turned out that
there exists an almost linear relation between the content of
the iron high-spin state of the complex in the ferric form and
the effective CO stretch frequency n'(CO) at room
temperature as shown in Fig. 10(A) (Jung et al., 1996b).
Complexes with a small high-spin state content tend to have
a CO stretch mode at higher frequencies. The high-spin
complex of P450s has been regarded as representing an
apolar and the low-spin complex a polar heme environment.
The increase of the CO stretch frequency when the heme
environment becomes more polar is unexpected. Usually,
for protein-free heme carbonyl complexes the inverse
behavior is observed. Polar solvents induce a decrease of
the CO stretch mode wavenumber due to a direct interaction
between the solvent molecules and the CO ligand. Therefore, an indirect effect of the polarity of the heme
environment in P450cam should exist. It was proposed that
the electrostatic field of the water molecules present in the
low-spin state might partially compensate the supposed
positive electrostatic potential in the I-helix groove around
Thr252 near the CO ligand (Jung et al., 1996b). Hydrogen
bonding between the CO ligand and amino acid residues in
the vicinity of the heme has been excluded from pHdependence studies of the CO stretch mode in camphorbound P450cam (Schulze et al., 1994b). Hydrogen bonding
would be indicated by an population exchange between
subconformers when the pH is changed, as seen for
myoglobin (Muller et al., 1999) or horseradish peroxidase
(Barlow et al., 1976). Such effects have not been observed
for P450cam in the presence of (1R)-camphor, although a
small pH-dependent shift of the n(CO) by about 1.5 cm1
with a pKa of about 6.2 is visible. This pKa is in agreement
J. Mol. Recognit. 2000;13:325351
341
Figure 10. Relation of the CO ligand stretch mode frequency in cytochrome

P450cam with two further functional important parameters. (A) Correlation of the
apparent CO stretch mode frequency in the CO complex of various substrate
complexes of P450cam (Fig. 8) with the high-spin state content induced by the
substrates in the Fe3 form (Jung et al., 1996b). The inset indicates the heme iron
coordination sphere in the Fe3 form for the substrate-free protein with 100% lowspin-state population and the (1R)-camphor-bound protein with 100% high-spin
state population (Poulos et al., 1987). The intermediate high-spin state contents
for the various substrate complexes can be explained by the mobility of the
substrate inside the heme pocket, which allows a certain accessibility of the heme
pocket for water molecules (Raag and Poulos, 1991; Jung et al., 1996b; Schulze et
al., 1997). (B) Correlation of the apparent CO stretch mode frequency in the
cytochrome P450camCO complex in the presence of various substrates (Jung et
al., 1996b) with the percentage amount of dioxygen which is converted in the
reaction cycle of P450cam to hydrogen peroxide (%H2O2; uncoupling side
reaction) in activity studies using the complete monooxygenase system (Kozin
and Hui Bon Hoa, 2000, in preparation). (1R)-camphor (1); (1S)-camphor (2);
camphor-N-methylimine (3); camphor-oxime (4); fenchone (5); norcamphor (6);
camphane (7); endo-borneol propyl ether (8).
with the pKa of 6 observed for the high-spin/low-spin

equilibrium.
P450cam CO ligand stretch mode and charge donation
from the proximal cysteinate ligand. Since the discovery
that a negatively charged sulfur of a cysteine is the fifth
ligand, the unique function of P450 to activate molecular
oxygen has been seen in the charge donation from the sulfur
to the dioxygen ligand. Quantum chemical calculations of
the O2 as well CO complexes of porphyrin iron complexes
with either imidazole or sulfur clearly indicated that the
charge donation to O2 or CO is more pronounced for the
negative sulfur ligand than for imidazole. Therefore, a lower
CO or O2 stretch mode frequency should be expected for
P450s compared to myoglobins or hemoglobins. The
experimental studies were, however, disappointing at first,
revealing CO stretch modes in the same wide frequency
range as also observed for heme proteins with histidine as
the fifth ligand. For heme proteins and their mutants and for
iron porphyrin complexes with imidazole as fifth ligand, an
inverse linear correlation between the CO stretch mode
frequency and the FeCO stretch mode frequency has been
observed from resonance Raman and infrared studies, as
already discussed above (Fig. 6). For weak proximal
ligands, as observed in some model complexes and
cytochrome c oxidase, or in the absence of the proximal
ligand, the line is shifted to higher frequencies. Such an
inverse relation between n(CO) and n(Fe-CO) has also been
shown for various substrate complexes of P450cam
(Legrand et al., 1995). However the line is shifted to lower
n(CO) values compared with the line for myoglobins and
hemoglobins. So, the stronger charge donation from the

cysteine proximal ligand is not directly seen in the absolute
values of n(CO) but in the shifted line.
P450cam CO ligand stretch mode and relation to
function. CO rebinding after flashphotolysis has recently
been carried out by Contzen and Jung (1998) for several
substrate complexes of P450cam using step-scan timeresolved FT infrared spectroscopy. The CO rebinding is
very fast for substrate complexes which are characterized by
a small high-spin content, by a fast entry of water molecules
into the heme pocket (Schulze et al., 1997) and by a high
frequency of the CO stretch mode in the CO complex (Jung
et al., 1996b). Studies on the P450cam substrate complexes
using FTIR and several other methods revealed a consistent
picture that substrates missing or having disturbed hydrophobic contacts to Val295, Asp297 and Val247 (Fig. 9) are
more mobile in the active center (Contzen et al., 1996;
Schulze et al., 1996; Schlichting et al., 1997), allow higher
access of the protein for water molecules (Schulze et al.,
1997), induce a high compressibility or volume fluctuation
of the protein (Jung et al., 1995), cause a high apparent CO
stretch mode in the CO complex (Jung et al., 1996b) and
show a prefered formation of hydrogen peroxide instead of
the substrate hydroxylation. Figure 10(B) shows a correlation between the apparent CO stretch mode frequency at
room temperature for various P450camCO substrate
complexes with the percentage of consumed dioxygen
which is converted to hydrogen peroxide in activity studies.
The H2O2 formation represents a side reaction in the P450
reaction cycle after O2 has bound to the heme iron. It
J. Mol. Recognit. 2000;13:325351
342
C. JUNG
competes with the expected formation of the iron-oxo

intermediate which hydroxylates substrates (Lewis, 1996).
Whether the n(OO) stretch vibration mode correlates with
the H2O2 formation similarily to the CO stretch mode is not
known. FT infrared studies of the O2 complex of P450 have
not been carried out because of the instability of the
complex and its difficult handling with FTIR. Few
resonance Raman studies exist which revealed the OO
stretch mode for (1R)-camphor-bound P450cam at
1140 cm1 (Bangcharoenpaurpong et al., 1986; Hu et al.,
1991; Macdonald et al., 1999) which is in the region
expected for a bent-on FeOO geometry (Schlichting et
al., 2000).
CO ligand stretch modes of steroid converting P450s.
Results of FTIR studies on other P450s are not so
straightforward. Correlations as seen in Figs 6 and 10(A)
fail to hold within the various substrate complexes of
P450sccCO (Tsubaki et al., 1992; Table 4). The substratefree protein (almost low-spin) and the cholesterol-bound
protein (almost high-spin), show a similar CO-stretch
frequency at around 1952 cm1. Equally surprising are
recent infrared studies on P45015b, which is completely
low-spin in the substrate-free as well as substrate-bound
state (deoxycorticosterone; Simgen et al., 2000). In the
absence of the substrate, several CO stretch modes exist
with a major band at about 1946 cm1 (Table 4). The
population of the lower-frequency mode at about
1936 cm1 is strongly increased when deoxycorticosterone
is bound. In contrast to substrates of P450cam, the steroid
substrates for P450scc and P45015b are rather bulky.
Therefore steric constraints may be more relevant in these
P450s than the compensating electrostatic effect of water
molecules discussed for P450cam.
NO synthase. The biogenesis of nitric oxide is catalyzed by
NO synthases (NOSs) which form L-citrulline and NO
through a stepwise NADPH and O2 dependent oxidation of
L-arginine (Stuehr, 1999). The first FTIR study of the CO
stretch vibration mode of the inducible nitric oxide synthase
oxygenase domain (iNOSox) has recently been reported by
Jung et al. (2000) for the temperature range from 20 to
298K. iNOSox in the absence of arginine reveals a
temperature-dependent equilibrium of two major conformational substates with CO stretch bands centered at about
1945 and 1954 cm1. This behavior is not qualitatively
changed when tetrahydrobiopterin (H4B) is bound. Arginine
binding changes the spectrum significantly by formation of
a sharp CO stretch mode band at about 1904 cm1,
indicating the formation of a hydrogen bond to the CO
ligand. It is suggested that the arginine itself may donate the
hydrogen. It is an open question whether the proposed
hydrogen bond is also present for the dioxygen complex
because recent resonance Raman measurements of the O
O stretch vibration, observed at 1135 cm1, did not reveal
an influence of the arginine binding (Couture et al., 2000).
Chloroperoxidase. Chloroperoxidase isolated from Caldariomyces fumago has been shown by various methods to
have a cysteinate proximal heme iron ligand (Dawson and
Sono, 1987; Sundaramoorthy et al., 1993). This enzyme
catalyzes the hydrogen peroxide-dependent oxidation of I,
Br and Cl with the resulting formation of a carbon
halogen bond with various compounds. The vibrational

properties of the heme complex have mainly been studied
by resonance Raman spectroscopy (Hu and Kincaid, 1993).
Infrared data are reported by OKeefe et al., (1978), giving a
CO stretch mode at 1942 cm1 for pH 3 and at 1958 cm1
for pH 6. Scholl (1991) has shown that the infrared spectrum
of the CO ligand stretch mode is very complex, with several
bands between 1930 and 1970 cm1 which are strongly
dependent on the pH, temperature and pressure. Because the
pH dependence is completely different from P450cam one
may conclude that the interactions with CO on the distal
side are very different. Indeed, the crystal structure
(Sundaramoorthy et al., 1993) reveals a glutamic acid in
the active side which can induce hydrogen bonding to the
CO ligand. In this regard, chloroperoxidase is similar to
other peroxidases which have a distal histidine hydrogen
bonding to the CO ligand.
Recognition phenomena in non-heme metal centers in
proteins
Ca2 binding. Various proteins have metal centers which
stabilize the protein structure (Jackson et al., 1991) or have
other functions (Ermler et al., 1998). The effect of Ca2
binding on the secondary structure monitored on the amide I
band has recently been reviewed by Surewicz and Mantsch
(1996) and Arrondo and Goni (1999). Metal ions are held in
place by coordinating histidines or carboxylate side chains
from glutamate or aspartate. Nara et al. (1994) have shown
for pike parvalbumin, which belongs to the large class of
Ca2 binding proteins, that the asymmetric stretch mode of
COO can be used as marker band for metal binding. In
neutral D2O, the bCOO of aspartate shows a band at
1584 cm1 and the COO group of glutamate at
1567 cm1. These signals are down-shifted to 1547 and
1551 cm1, respectively, when coordinating Ca2 in a
didentate form. Ca2 binding in horseradish peroxidase has
also been monitored using the antisymmetric stretch
vibration observed at 1554 cm1 (Kaposi et al., 1999).
Dzwolak et al. (1999) used this spectral marker to study the
effect of Ca2 on the barostability of bovine a-lactalbumin.
The symmetric stretch vibration observed at about
1388 cm1 has also been shown to be affected by Ca2
binding to a-lactalbumin and lysozyme (Mizuguchi et al.,
1997).
Hydrogenases. Hydrogenases form a family of enzymes
found in various microorganisms which split molecular
hydrogen (Ermler et al., 1998). Two types of classes of
hydrogenases are known: the Ni/Fe hydrogenases where Ni
and Fe form a binuclear center and the Fe hydrogenases
which contain only iron in the active center. In 1994 Bagley
et al. reported infrared spectra of Ni/Fe hydrogenases,
revealing unusual signals at 1944 cm1 (strong) and at 2081
and 2093 cm1 (weaker) in the oxidized form which shift to
1929, 2060 and 2069 cm1 when reduced with ascorbate
under CO atmosphere. Van der Spek et al. (1996) have
shown that such signals are exclusively observed for Ni/Fehydrogenases and Fe-hydrogenases and not seen in other
non-metal-hydrogenases or various ironsulfur proteins.
Based on 13C and 15N labeling of Ni/Fe-hydrogenase from
J. Mol. Recognit. 2000;13:325351
343
Figure 11. CO stretch mode infrared spectra of cytochrome P450camCO. In the absence
(top) and in the presence of (1R)-camphor (buttom) as a function of the temperature
(left), of the hydrostatic pressure (middle) and for selected times after a pressure jump
(fast release from 200 to 40 MPa) (right). Experimental conditions are described in
Schulze et al. (1994b), Scholl (1991) and Jung et al. (1992a, 1996b).
Desulfovibrio gigas, Happe et al. (1997) have found that

two internal cyanide groups and one internal CO molecule
are bound to the Fe ion in the NiFe binuclear center, which
causes the infrared bands at 2093, 2083 and 1945 cm1,
respectively.
protein unfolding are discussed by Surewicz and Mantsch

(1996).
Substrate binding. Substrate binding and recognition by

the enzyme can be followed by FTIR in different ways. One
can monitor a spectroscopic probe molecule near the
substrate binding site as demonstrated above for P450 or
the substrates itself have infrared active modes of high
diagnostic value which are affected by the binding. For
example triosephosphate isomerase catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde 3-phosphate (GAP). The carbonyl group of
unbound DHAP appears at 1730 cm1 and is shifted to
1715 cm1 when bound to the enzyme. This shift indicates
that the carbonyl group of the substrate is more polarized in
the bound state by forming electrostatic or hydrogen
bonding interactions with the active site histidine His95
(Zhan et al., 1999). Increased polarization is required for the
functionally important proton shuttle.
The effect of substrate and ligand binding on the
secondary structure can also be followed on the amide I
band. Usually, data about global structural changes are
obtained which are less informative for specific recognition
processes. Further examples of monitoring substratespecific infrared probes and effects of substrate binding on
FTIR spectroscopy has been used to study the dynamics of

proteins and proteinligand interactions. Two approaches
are applied: (i) analysis of the change of the population of
conformational substates with changing the temperature
(and/or pressure); and (ii) following the ligand rebinding
process or conformational relaxation using time-resolved
FTIR. Most of the studies have been performed on heme
proteins.
Study of proteinligand dynamics using lowtemperature, high-pressure and time-resolved FTIR
Static low-temperature and high-pressure studies of

heme protein CO complexes. With lowering of the
temperature (and/or increasing the pressure) a population
exchange between conformational substates, reflected in the
different CO stretch modes, is observed. This exchange
generally stops rather sharply when passing the temperature
range between 200 and 170K, that is the liquid/glass phase
transition of the solvent/co-solvent mixture (Jung et al.,
1996a) where the population equilibria freeze-in (Mayer,
1994a) or the substate transitions are strongly damped
because of the high viscosity. Figure 11 shows the
temperature and pressure dependence of the CO stretch
mode infrared spectra of P450cam in the absence and in the
J. Mol. Recognit. 2000;13:325351
344
C. JUNG
Figure 12. Difference infrared spectra (`light minus dark') for photodissociated cytochrome P450camCO in the presence of two different
substrates. (1R)-camphor and 1-iodoadamantane at 20K; 50 mM potassium phosphate buffer, pH 7, 60% (v/v) glycerol, CaF2 windows, 100 m
spacer, c(P450) 1 mM, photodissociation induced by the NdYAG laser
with 532 nm (Jung, unpublished).
presence of the substrate (1R)-camphor. In the absence of a

substrate, decreasing temperature shifts the substate equilibrium to the lower-frequency mode while increasing
hydrostatic pressure favors the substate with the higherfrequency mode (Jung et al., 1996a). The kinetics of the
substate transitions has been studied with the pressure jump
(fast release from 200 to 40 MPa) technique (Frauenfelder et
al., 1990; Scholl, 1991; Jung et al., 1992a). When P450cam
recognizes the substrate (1R)-camphor, the number of
substates is significantly restricted. However, low-temperature and high-pressure studies reveal that at least two
substates are present whose interconversion can be followed
by the pressure jump technique (Schulze et al., 1994b;
Scholl, 1991, Jung et al., 1992a).
The CO stretch mode frequency and width of the CO
stretch bands are also affected by the temperature
(pressure). The frequency is shifted by several wavenumbers (5 cm1 from 298 to 200K) with almost no change or
a change with a much smaller slope when further cooled
down to 20K (Jung and Marlow, 1987; Schulze, 1997). In
myoglobin, for example, a frequency shift to higher values
is observed for mode Ao and mode A1 (Ansari et al., 1987).
Mode A3 tends to shift down with cooling (Mayer, 1994b).
However, in hydrated films a sharp breakdown of the
changes in population and frequency at around 180 200K is
not observed (Mayer, 1994b) so water as an important
component in the protein strongly enhances the internal
mobility of the protein (Fischer and Verma, 1999) resulting
in a sharp change of the infrared parameters at around 180K.
In P450cam, in the presence of various substrates, a
decrease of the CO stretch mode frequency is observed on
cooling from 298K to about 180K with a much smaller slope
below this temperature (Schulze et al., 1994b; Schulze,
1997). However for substrate-free P450cam, which is
known to have more water in the protein, the higherfrequency CO stretch mode at about 1954 cm1 shifts up
with cooling. For this and also for the mode at around
1963 cm1, an inverse effect of hydrostatic pressure and
glycerol (interpreted as osmotic pressure) on the frequency

has been observed (Jung et al., 1996a). The inverse
influence of hydrostatic and osmotic pressure on the CO
stretch modes indicates that these higher-frequency modes
reflect subconformations which are more accessible for
water.
Thus, the effect of temperature and pressure on the CO
stretch modes is determined by a balance of several physical
effects such as volume contraction which forces hydrogen
bonding (Demmel et al., 1997), dielectric constant (Jung et
al., 1996a) and pH changes of the solvent (Schulze et al.,
1994a, 1997), which may influence the local environment of
the CO ligand. This should be considered when recognition
phenomena are studied using the CO stretch mode.
CO ligand flashphotolysis. Most of the studies use the
heme Soret electronic absorption band to follow the
rebinding process. The Soret band is, however, not sensitive
enough to distinguish between the relaxation behavior of the
conformational substates and its possible influence of
substrates. FT infrared spectroscopy can, however, do this
and has advantages for following structural changes in
different parts of the protein.
A simplified model describes the photodissociation and
rebinding in four main steps. After breaking the ironligand
bond (step 1) by light absorption of the heme, the ligand
moves to a docking site within the heme pocket (step 2).
Then it migrates through the protein (step 3) (probably from
one site to another) before it escapes to the solvent (step 4).
Rebinding from the solvent might follow the same pathway
or others. At low temperature (<160K) the photodissociated
CO ligand, docked to sites near the heme, cannot leave the
heme pocket. Only geminate rebinding occurs. At very low
temperatures (<60K) the geminate rebinding is so slow that
one can easily observe the CO stretch mode of the
dissociated CO ligand (the so-called B-state) in the infrared
difference spectrum to the bound state (the so-called Astate). Figure 12 shows the light minus dark infrared
J. Mol. Recognit. 2000;13:325351
difference spectra of P450cam for two different substrate

complexes as a typical example. Analogous spectra for
myoglobin and various mutants are given by Braunstein et
al. (1993).
The CO stretch modes of the B-states are observed in the
range between 2100 and 2150 cm1 (Fig. 12) near the
stretch mode of the free CO (2143 cm1). Usually, multiple
A-states correspond to multiple B-states (Mourant et al.,
1993; Jung et al., 1992a). The integral intensity of the total
B-state band spectrum is approximately 1730 times weaker
than that of the A-state signals. The concrete value of the
CO stretch mode frequency of the B-state is determined by
the microenvironment of the docking site which polarizes
the CO dipole. The B-states have been studied in detail on
wild-type myoglobin (Mourant et al., 1993; Lim et al.,
1995a,b, 1997; Nienhaus et al., 1994), various myoglobin
mutants (Braunstein et al., 1993), on hemoglobin (Lim et
al., 1995a), on leghemoglobin (Stetzkowski et al., 1985), on
cytochrome P450 (Jung et al., 1992a) and on NO synthase
(Jung et al., 2000). Using an ultrafast time-resolved infrared
spectrometer Lim et al. (1995a,b) have found that the
photodissociated CO molecule is hosted in a rotationally
constrained environment. From measurements of the
polarization anisotropy decay with femtosecond time
resolution, Lim et al. (1997) concluded that the two Bstates in myoglobin observed with FTIR may correspond to
the same docking site but with a rotated CO molecule
showing rotational time constants in the range between 200
and 500 fs (Anfinrud et al., 1999). Molecular dynamics
simulations for sperm whale myoglobin by Meller and Elber
(1998) have assigned the docking site to be above the heme
and between Leu29, Val68 and Ile107 in agreement with
sites identified in the crystal structure of the photodissociated myoglobin (Hartmann et al., 1996). Other docking
sites in horse heart myoglobin and mutants have recently
been detected (Chu et al., 2000; Brunori et al., 2000).
The CO stretch signals monitor local events. To understand how the apparent local changes are accompanied by
structural changes in the whole protein, further spectroscopic probes are needed. Infrared spectroscopy is an ideal
method to follow simultanously the broad spectral range
where various signals of the different absorbing groups
appear. An example has been discussed above for oxidases
(Puustinen et al., 1997). Static difference infrared spectra
between the CO bound and deoxy hemoglobin in D2Odialyzed buffer indicated that distinct changes can be seen
also in the amide I and amide II band region (Gregoriou et
al., 1995). These changes are very small compared with the
absolute intensity of the amide bands. A very accurate
subtraction of the reference sample is required, which is a
very tricky task. This problem does not exist when the stepscan time-resolved FTIR spectroscopy is used. This technique has been applied first to myoglobin (Plunkett et al.,
1995) and hemoglobin (Hu et al., 1996). Contzen and Jung
(1998) used this technique to study the effect of substrate
binding on the rebinding kinetics of the flashphotolyzed CO
ligand.
Figure 13 shows a typical step-scan infrared difference
spectrum for selected times with P450cam as an example.
Structural changes in the amide I region appear, indicating
small changes in the secondary structure, but changes of the
heme or other side groups of the protein may also be
345
Figure 13. Time-resolved absorption difference infrared spectra

(`light minus dark') for the rebinding of the ash-photolyzed CO
ligand of (1R)-camphor-bound cytochrome P450cam taken with
the step-scan time-resolved FTIR spectroscopy. 50 mM potassium
phosphate D2O buffer, pD 7; 50% (v/v) glycerol-d3,
c(P450) = 0.79 mM, 23.7 mM (1R)-camphor (Contzen and Jung,
1998). The time trace at a specic mirror position is shown in Fig.
1. The peak assignment follows Table 2 and the explaination
given in the text for salt links.
considered. The appearance of the signal around 1719cm1

is outside the crowded amide I region and is therefore more
specific and is assigned to a change in a salt link between the
heme 7-propionate and Asp297 and Arg299. A change in
the region between 1724 and 1713cm1 has also been
observed in infrared difference spectra induced by photoreduction and assigned to the salt bridge between the 6propionate of the heme and Arg112 and His355 (Contzen
and Jung, 1999). This change in the salt link is not observed
in the substrate-free protein, indicating that the substrate
binding affects not only the distal side structure near the
heme ligand but may also have a more global effect. In
addition, simultaneous monitoring the infrared signals,
reflecting different parts of the protein, may not show the
same kinetics which could pinpoint to a different dynamic
behavior of the protein substructures (Contzen and Jung,
1998).
Light-induced proton translocation and O2 evolution.
Uncovering the mechanism of proton translocation in
transmembrane pumps has benefited significantly from
infrared studies. Bacteriorhodopsin is a light-driven proton
pump. Proton translocation is the consequence of the
photoisomerization of the retinal chromophore (Oesterheld,
1998). The photocycle has various intermediates. The most
interesting point is to understand the structural differences
between these intermediates. Using light-induced infrared
difference spectroscopy coupled with low-temperature and
time-resolved techniques, isotope labeling and site-directed
mutagenesis, many groups (Tyr, Trp, Asp, Pro) involved in
proton movement have been identified (Rothschild et al.,
J. Mol. Recognit. 2000;13:325351
346
C. JUNG
1992; Maeda et al., 1997; Hessling et al., 1997).

Photosynthetic oxygen evolution takes place on the
electron-donor side of photosystem II (PS II). The catalytic
center consists of a tetranuclear Mn cluster where two water
molecules are oxidized in a light-driven cycle of five
intermediates to form one dioxygen and four protons. Using
light-induced difference infrared spectroscopy various
amino acid side chain groups have been identified to be
involved in the reaction. For example, using 15N-labeled
spinach and PS II core complexes from Synechocystis cells
Noguchi et al., (1999) have assigned CH stretch and N
H modes of histidine (Table 3).
Although proton translocation and O2 evolution are not
within the scope of this review, it is important to mention
these studies because many infrared signals of amino acid
side chains have been assigned, which is very helpful in
understanding corresponding signals observed in protein
ligand recognition.
CONCLUDING REMARKS
chains whereby the carboxylate groups have a high

diagnostic value for salt bridges. Cofactors are accessible
to study by their specific vibrational modes. The stretch
mode of heme ligands may be used to probe the active
center in heme proteins. The stretch mode of the CO ligand
is a sensitive marker for the polarity of the heme pocket and
may also be used as a probe molecule for the study of the
protein dynamics. It is possible to study water molecules
bound to the protein. Because FTIR spectroscopy can be
combined with time-resolved, low-temperature and highpressure techniques, a broad field for studies of the
dynamics of the folding and unfolding of the protein
structure, and of the influence by ligand and substrate
binding is opened. FTIR spectroscopy is a powerful method
which can contribute significantly to analysis of recognition
phenomena when combined with isotope labeling, sitedirected mutagenesis and complemented with crystallography and further spectroscopic methods.
Acknowledgements
FTIR spectroscopy is one of the few methods which can

probe various local and global structural properties of
proteins and is therefore appropriate to study recognition
phenomena. The secondary structure of the protein can be
studied using the amide I band. Local structural changes can
be analyzed by the specific signals of the amino acid side
The former and present Ph.D. students Jorg Contzen, Heike Schulze,
Nathalie Legrand, Corinne Mouro and Eric Deprez from the authors and
cooperating laboratories are gratefully acknowledged for their work on
cytochrome P450. The author thanks Rebecca Wade for carefully reading
the manuscript and for many helpful comments.
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JOURNAL OF MOLECULAR RECOGNITION

J. Mol. Recognit. 2000;13:325351

Insight into protein structure and proteinligand

the problem of different recognition phenomena. How the

* Correspondence to: C. Jung, Max-Delbruck-Center for Molecular Medicine,

Copyright # 2000 John Wiley & Sons, Ltd.

In contrast to the dispersive spectrometer, where the

Figure 1. Principle of the experimental set-up of Fourier transform infrared

The result of the data acquisition is a digitized interferogram

Ne laser light. This interferogram is Fourier transformed

FTIR AND PROTEINLIGAND RECOGNITION

Time-resolved experiments are triggered or initiated by

applications for time-resolved measurements. At each

Most of the infrared bands of proteins are relatively broad.

genous broadening is always present. The band shape must

FTIR AND PROTEINLIGAND RECOGNITION

mathematical description for an ensemble of bands which

INFRARED SIGNAL ASSIGNMENT

the amide I band appearing between 1600 and 1700cm1

Arg side chain

composition obtained from CD measurements of the

Chirgadze et al. (1975) for D2O solutions and by

FTIR AND PROTEINLIGAND RECOGNITION

Table 3. Amino acid side chain infrared absorption signals

The SH sulfhydryl group of cystein absorbs between

Roepe et al. (1987)

Maeda et al. (1992)

position depends on the protonation state of the nitrogen.

originate from porphyrin or its substituents (Schlereth and

orbitals would best describe the structure.

FTIR AND PROTEINLIGAND RECOGNITION

Cyanide. CN can bind to either ferric or ferrous heme

molecules to the protein is mediated by hydrogen bonding

Azide. Free azide is linear with symmetric NN bonds.

Environmental effects. The concrete value of the ligand

al., 1999) is in agreement with this observation. This crystal

and forming a hydrogen bond.

FTIR AND PROTEINLIGAND RECOGNITION

contact, does not seem to exist.

vis hemoglobin. The CN stretch mode for the Fe(III)

Figure 6. Correlation of the iron-bound CO and the FeCO

than the CO ligand because it approches the iron as HCN

hemoglobin >8 cm1), which has been explained with a

FTIR AND PROTEINLIGAND RECOGNITION

play a role in the proton translocation and therefore for the

CN is released from the complex upon photoreduction and

Bohm et al. (1976)

Tsubaki et al. (1992)

1937.5 (13.4, 0.156)

Simgen et al. (2000)

Tsubaki et al. (1992)

Simgen et al. (2000)

Jung and Marlow (1987)

OKeefe et al. (1978)

1941.4 (10.2, 1.00)

Copyright # 2000 John Wiley & Sons, Ltd.

Jung and Marlow (1987)

Jung et al. (1996b); Legrand et al.

J. Mol. Recognit. 2000;13:325351

FTIR AND PROTEINLIGAND RECOGNITION

Endo-borneol allyl ether

Jung et al. (1996b)

Jung et al. (1996b); Legrand et al.

1942.9 (19.4, 0.29);