Photosynth Res (2009) 101:157–170

DOI 10.1007/s11120-009-9439-x

REVIEW

Fourier transform infrared (FTIR) spectroscopy

Catherine Berthomieu Æ Rainer Hienerwadel

Received: 18 February 2009 / Accepted: 15 May 2009 / Published online: 10 June 2009

Springer Science+Business Media B.V. 2009

Abstract Fourier transform infrared (FTIR) spectroscopy often used to identify the chemical groups. Studies on model
probes the vibrational properties of amino acids and cofac- compounds and the increasing use of theoretical chemistry
tors, which are sensitive to minute structural changes. The for normal modes calculations allow us to interpret the IR
lack of specificity of this technique, on the one hand, permits frequencies in terms of specific structural characteristics of
us to probe directly the vibrational properties of almost all the chemical group or molecule of interest. This review
the cofactors, amino acid side chains, and of water mole- presents basics of FTIR spectroscopy technique and provides
cules. On the other hand, we can use reaction-induced FTIR specific important structural and functional information
difference spectroscopy to select vibrations corresponding to obtained from the analysis of the data from the photosystems,
single chemical groups involved in a specific reaction. using this method.
Various strategies are used to identify the IR signatures of
each residue of interest in the resulting reaction-induced Keywords Fourier transform infrared spectroscopy

FTIR difference spectra. (Specific) Isotope labeling, site- Isotope-edited infrared spectroscopy
Metal–ligands

directed mutagenesis, hydrogen/deuterium exchange are Water molecules

List of abbreviations
Special Issue of Photosynthesis Research ‘‘Basics and Applications of ATR Attenuated total reflection
BiophysicalTechniques in Photosynthesis and Related Processes’’,
edited by Messinger, Alia and Govindjee. BChl Bacteriochlorophyll
CVD Chemical vapor deposition
C. Berthomieu (&) ENDOR Electron nuclear double resonance
CEA (Commissariat à l’ Energie Atomique), Laboratoire des ESEEM Electron spin echo envelope modulation
Interactions Protéine Métal, DSV (Direction des Sciences du
Vivant), Institut de Biologie Environnementale et FTIR Fourier transform infrared
Biotechnologie, Service de Biologie Végétale et Microbiologie IR Infrared
Environnementales (DSV/iBEB/SBVME), CEA-Cadarache, P700 Primary electron donor of PSI
UMR 6191 Centre National de la Recherche Scientifique PSI Photosystem I
(CNRS)-CEA (Commissariat à l’ Energie Atomique)-Université
Aix-Marseille II, 13108 Saint Paul-lez-Durance Cedex, France PSII Photosystem II
e-mail: [email protected] QA, QB Primary and secondary electron acceptor
quinones of photosynthetic RCs
R. Hienerwadel RC Reaction center
Aix-Marseille Université, Laboratoire de Génétique et de
Biophysique des Plantes, Faculté des Sciences de Luminy, TyrD, TyrZ The two redox active tyrosines of PSII
Direction des Sciences du Vivant, Institut de Biologie WT Wild type
Environnementale et Biotechnologie, Service de Biologie
Végétale et Microbiologie Environnementales (DSV/iBEB/ Introduction
SBVME), UMR 6191 Centre National de la Recherche
Scientifique (CNRS)-CEA (Commissariat à l’ Energie
Atomique)-Université Aix-Marseille II, 13288 Marseille Infrared (IR) or Fourier transform infrared (FTIR) spec-
cedex 9, France troscopy has a large application range, from the analysis of

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158 Photosynth Res (2009) 101:157–170

small molecules or molecular complexes to the analysis of Principle and methodology of infrared spectroscopy
cells or tissues. The imaging of tissues is one of the recent and FTIR difference spectroscopy
developments of infrared spectroscopy, taking advantage
of infrared microscopy and of the use of synchrotron IR Infrared spectroscopy
radiation. It is used for the mapping of cellular components
(carbohydrates, lipids, proteins) to identify abnormal cells Infrared spectroscopy probes the molecular vibrations.
(Levin and Bhargava 2005; Petibois and Déléris 2006). Functional groups can be associated with characteristic
FTIR spectroscopy has also been increasingly applied to infrared absorption bands, which correspond to the funda-
the study of proteins. This concerns the analysis of protein mental vibrations of the functional groups (Colthup et al.
conformation, protein folding, and of molecular details 1975; Griffith and de Haseth 1986). For a nonlinear mol-
from protein active sites during enzyme reactions using ecule with N atoms, there are 3N-6 vibrational motions of
reaction-induced FTIR difference spectroscopy (Siebert the molecule atoms, or 3N-6 fundamental vibrations or
and Hildebrandt 2008). normal modes. A normal mode of vibration is infrared
FTIR difference spectroscopy has been widely applied active (i.e., it absorbs the incident infrared light) if there is
in photosynthesis research and related areas. This approach a change in the dipole moment2 of the molecule during the
gives complementary information to the three-dimensional course of the vibration. Thus, symmetric vibrations are
structural data obtained by X-ray diffraction (or Nuclear usually not detected in infrared. In particular, when a
Magnetic Resonance, NMR). The analysis of active sites in molecule has a center of symmetry, all vibrations which are
proteins by means of reaction-induced FTIR difference symmetrical with respect to the center are infrared inactive.
spectroscopy gives information on minute structural In contrast, the asymmetric vibrations of all molecules are
changes, hydrogen-bonding interactions, and proton trans- detected. This lack of selectivity allows us to probe the
fer reactions, which are often beyond the sensitivity of X- properties of almost all chemical groups in one sample, and
ray diffraction analyses. Moreover, time-resolved tech- notably of amino acids and water molecules which can
niques, with time resolution now up to the femtosecond hardly be observed by other spectroscopic techniques.
range (Di Donato et al. 2008) allow structural changes to Strong IR absorptions are observed for groups with a
be observed for protein active sites ‘‘at work’’. permanent dipole (i.e., for polar bonds). As such, the car-
In photosynthesis, this approach has given information bonyl groups of the polypeptide backbone contribute lar-
of prime interest concerning the cofactor-protein interac- gely to the infrared absorption spectra of proteins.
tions (Nabedryk 1996; Breton 2001; Berthomieu and In the mid-infrared region (4,000–1,000 cm-1), two
Hienerwadel 2005; Noguchi and Berthomieu 2005), as well main types of vibrations are observed: vibrations along
as proton transfer routes in bacterial reaction centers chemical bonds, called stretching vibrations (m), which
(Nabedryk and Breton 2008). It now plays a central role in involve bond-length changes; and vibrations involving
the detailed analysis of the oxygen evolving complex of changes in bond angles, and notably bending vibrations
Photosystem II (Chu et al. 2001; Noguchi 2007; Debus (d—in plane, p—out of plane).
2008). The stretching vibrations can be modeled using the
While this approach suffers from several limitations, it harmonic oscillator model (Fig. 1), in which a chemical
delivers unique information by addressing directly the bond is represented by two point masses linked by a spring.
properties of cofactors, amino acids, and water molecules, The bond strength (or molecular force field) is the spring
with very high sensitivity to structural parameters and tenseness k and the point masses (m1 and m2) model the
electronic interactions. This justifies the experimental masses of the atoms or chemical groups involved in the
efforts that have been made to optimize its use and the bond. The oscillation frequency m is given by the equation3:
interpretation of the data. pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
m ¼ ð1=2pcÞ ðkðm1 þ m2 Þ=m1 m2 Þ:
In the following, we briefly present the principles of
infrared spectroscopy and describe the development of The vibration frequency m thus depends on the bond
experimental approaches to identify and analyze IR sig- strength, with higher frequencies for triple or double bonds
natures from active sites in proteins by reaction-induced as compared to single bonds. This is illustrated in Fig. 1
FTIR difference spectroscopy. We describe the methodol- for CO bonds. A consequence of the dependence of
ogy to obtain reliable spectra and interpretations, and show stretching mode frequencies on the bond strength is that the
typical examples of specific information brought by this 2
Dipole moment: when a positive charge ?z and a negative charge
technique in the study of photosystems.1
-z are separated by a distance d, the dipole moment l is equal to the
magnitude of the charge multiplied by the distance (l = zd). .
1 3
We do not present an exhaustive review of the FTIR literature on In the FTIR spectra, the infrared absorption is given as a function of
photosystems. the vibration frequency expressed in cm-1.

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Photosynth Res (2009) 101:157–170 159

In summary, the vibrational frequencies of a given

chemical group are expected in specific regions which
depend on the type of atoms involved and the type of
chemical bonds. Tables are available for the main chemical
groups, as well as for amino acid side chains (Venyaminov
and Kalnin 1990a; Socrates 1994; Barth 2000; Wolpert and
Hellwig 2006). Within these vibration regions, the fre-
quencies of the chemical groups are modulated by the
specific environment of the group. Therefore, to establish a
clear relationship between the infrared mode frequency and
the structural properties of a given residue, or when there
are no data available in the tables, it is necessary to perform
a detailed IR analysis on simplified model compounds in
different environments (solvents) and/or to analyze exper-
imental results using theoretical chemistry approaches.

Absorption of proteins in solution

The infrared absorption spectrum of proteins is dominated

by the absorption of the peptide bonds (Fig. 2a). The
m(C=O) modes of the peptide backbone contribute in the
1,680–1,620 cm-1 range and form the Amide I band, while
Fig. 1 Stretching vibrations can be approximately determined using the peptide m(CN) ? d(NH) modes contribute in the 1,560–
the harmonic oscillator model. The infrared absorption frequencies, v,
of these vibrations depend on the nature of the chemical bonds, 1,520 cm-1 range and form the Amide II band. The Amide
interactions with the environment (e.g., H-bonding) and relative I band of a protein depends on the tertiary structure of the
masses of the atoms involved in the vibration. Typical frequency- protein, with distinct m(C=O) mode frequencies for a-
shifts are indicated for m(CO) vibrations for these effects helices, turns, or b-sheet structures (Krimm and Bandekar

frequencies are very sensitive to the group environment,

the electronegativity of neighboring atoms or groups, or to
hydrogen bonding interactions. The involvement of one of
the atoms in a hydrogen bond will induce a weakening of
the bond strength, and thus a frequency downshift of the
stretching mode of the chemical group (Fig. 1). The sen-
sitivity of this method is high, and corresponds to changes
in bond length smaller than 0.2 Å (Deng and Callender
1999; Barth 2007). For a carbonyl group, formation of a
hydrogen bond induces downshifts up to 20 cm-1. The
inspection of stretching mode frequencies of chemical
groups, and specifically of carbonyl or carboxylic groups in
proteins, reveals information on fine structural details.
Formation or disruption of hydrogen bonding interactions
is one of those.
The vibration frequency m also depends on the mass of
the atoms involved in the vibration. This effect is illus-
trated in Fig. 1. Consequently, it is possible to specifically
alter a vibration frequency by isotope labeling of one of the
atoms involved in the vibration. Substitution of hydrogen Fig. 2 a Infrared absorption spectrum of thylakoid membranes
by deuterium has been widely used to identify and analyse enriched in Photosystem II in the mid-infrared region (continuous
groups with exchangeable protons. Specific isotope label- line), contribution from water molecules of the buffer (dotted line). b
Light-induced FTIR difference spectrum corresponding to TyrD
ing (13C, 15N, 18O, 2H) is also a very powerful approach to oxidation in Photosystem II. Negative bands correspond to the dark
identify the frequency of one specific group in a complex adapted resting state, while the positive bands correspond to the light-
infrared spectrum (see below). induced Tyr•D state (Hienerwadel et al. 1996)

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160 Photosynth Res (2009) 101:157–170

1986; Venyaminov and Kalnin 1990b), corresponding to illustrates a specific interest of infrared spectroscopy to
different hydrogen bonding patterns and electrostatic directly probe the protonation state of aspartate or gluta-
interactions of the peptide carbonyl groups in these struc- mate groups, and hence their involvement in proton
tures. The structural analysis of proteins thus largely transfer pathways, and to study their properties as metal–
involves IR spectroscopy, notably to inspect the effect of ligands in proteins.
temperature on protein folding (Hauser et al. 2008; Muk- Other amino acids, and notably tyrosine, arginine or
herjee et al. 2008) or the consequences of protein–protein histidine have typical vibrations, which have been useful in
interactions (Karyakin et al. 2007). identifying their contributions to infrared spectra and their
Water also contributes with two main bands in the mid- properties in various proteins. The hydrogen bonding
infrared: the intense OH stretching mode, m(OH), between properties of the two redox-active tyrosines of Photosystem
3,600 and 3,100 cm-1 and the HOH bending mode, II (PS II) have been analyzed in the reduced and oxidized
d(HOH), at & 1645 cm-1 (Fig. 2a). The m(OH) mode, states using FTIR difference spectroscopy (Hienerwadel
which is sensitive to hydrogen bonding properties of water et al. 1997, 2008; Noguchi et al. 1997; Berthomieu and
molecules, has been used to analyze water molecules at the Hienerwadel 2005). IR bands of histidine side chains are
active sites of bacterhiorhodopsin and Photosystem II (see sensitive to the protonation state of the imidazole side
Section ‘Properties and role of water molecules’). The chain as well as sensitive to metal-binding (Hasegawa et al.
absorption coefficient of the m(OH) mode is such that the 2000, 2002; Dupeyrat et al. 2004, and references therein).
IR detector is saturated in the 3,600–3,100 cm-1 region for These IR markers have been used to analyze histidine
an aqueous sample of even less than 10 lm path length. properties as a ligand of (bacterio)chlorophylls (Breton
Partially dehydrated samples must be used to identify the et al. 2002) or of Mn and the non-heme iron in Photosystem
role of specific water molecules at protein active sites. II (Hienerwadel and Berthomieu 1995; Noguchi et al.
Similarly, the absorption of an aqueous sample with 10 lm 1999; Yamanari et al. 2004; Kimura et al. 2005).
path length is above 1 absorption unit at 1,645 cm-1; thus,
it is too high to obtain reliable FTIR spectra. This is a FTIR difference spectroscopy
strong limitation for the analysis of samples in aqueous
solutions. Thin samples, with protein concentrations in the Sample preparation
millimolar range, or partially dried protein films have been
used for transmission infrared spectroscopy in water. Pro- The strong absorption of water and of the peptide bonds
tein structure analysis using the properties of the Amide I dominates the absorption spectrum of protein aqueous
band often involves samples in 2H2O: indeed, the samples. The samples thus consist of concentrated protein
d(2HO2H) mode contributes at &1,200 cm-1 and does solutions placed in measuring cells formed by two IR
not impair the analysis of the Amide I band at 1,680– transparent windows4 separated by a path length smaller
1,620 cm-1. than 10 lm. Dried films of bacterial reaction centers or
Amino acid side chains have a number of characteristic Photosystem I (PSI) have been used. For PSII, either a
normal modes in the mid infrared (Venyaminov and Kalnin pellet of PSII enriched membranes is squeezed between
1990a; Barth 2007). This is exemplified for aspartate or two windows, or concentrated solutions of PSII core
glutamate side chains. The protonated carboxylic groups complexes are partially dried on a window. The hydration
are characterized by the m(C=O) mode, which occurs above level can be controlled by the deposition of a small volume
1,680 cm-1 and is sensitive to hydrogen bonding interac- of a water–glycerol mixture at a defined water/glycerol
tions and to the environment polarity. For a hydrogen ratio in the sample chamber formed by the two tightly
bonded COOH group, the m(C=O) mode is downshifted by sealed IR windows (Noguchi and Sugiura 2002a).
up to 10 cm-1 upon H2O/2H2O exchange; this is often used In order to analyze an active site in a protein at the
as a diagnostic tool for such hydrogen bonding interactions molecular level, FTIR difference spectroscopy must be
in proteins. In contrast, the deprotonated carboxylate used. This approach couples the specific perturbation of the
groups have typical mas(COO-) and ms(COO-) modes at active site of interest with the recording of absorption
1,580–1,560 and 1,420–1,395 cm-1, respectively. These spectra of the sample before and after the perturbation. The
modes are also sensitive to the carboxylate environment. difference spectrum resulting from the subtraction of these
Upon carboxylate binding to a metal ion, the frequency two spectra contains contributions from the amino acids
difference (mas - ms) is usually altered as compared to the
typical frequency difference observed in solution. The
parameter (mas - ms) is smaller for a bidentate carboxylate 4
IR transparent materials must be used. For the mid-IR domain,
ligand and greater in the case of a monodentate ligand calcium fluoride is a material of choice for its moderate hygroscopic
(Deacon and Phillips 1980; Nakamoto 1997). This example character.

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Photosynth Res (2009) 101:157–170 161

and cofactors selectively perturbed. Such a difference form radicals of aromatic amino acids and notably tyrosine,
spectrum shows a large number of small signals which 4-methylimidazole, and phenol at low temperature in vitro
correspond to single vibrations (Fig. 2b). It is, therefore, of (Berthomieu and Boussac 1995; Berthomieu et al. 1998a;
great help to be able to perform the reaction repeatedly on Ayala et al. 2002).
the same sample, to average data from several cycles and
thus increase the signal to noise. Indeed, very small bands Electrochemically-induced FTIR difference spectroscopy
are observed in FTIR difference spectra (10-5–10-3 a.u.),
notably for large proteins such as PSII. FTIR-difference spectroscopy has been generalized to a
broader range of proteins, by the implementation of other
Light-induced FTIR difference spectroscopy means to trigger a reaction. Electrochemistry coupled to
FTIR difference spectroscopy allows us to probe all pro-
This approach was first developed on bacteriorhodopsin, teins with redox-centers. In the area of photosynthesis, it
using light as a trigger of the photochemical cycle was first used to study the IR properties of isolated redox
(Br ? M transition, Rothschild et al. 1981). The control of chlorophyll, quinone and heme cofactors. Indeed, a way to
the photo-induced reaction can be done (i) kinetically, by identify the contributions from cofactors in the complex
recording the spectrum during a short time after a light- FTIR difference spectra recorded with protein samples
flash or by using time-resolved FTIR difference spectros- involves the comparison with spectra of isolated model
copy; (ii) by controlling the sample temperature; (iii) by compounds. Thin path-length transmission cells were
using chemicals (electron donors and/or electron acceptors, developed to analyze model compounds in solvents
reducing or oxidizing agents) to poise a light-induced (Mäntele et al. 1988a, b; Bauscher et al. 1990), as well as
reaction intermediate. Time-resolved FTIR spectroscopy model compounds and proteins in aqueous solutions (Moss
involves rapid scan techniques, with 10–25 ms time reso- et al. 1990; Leonhard and Mäntele 1993; Berthomieu et al.
lution (Thibodeau et al. 1990) or step-scan FTIR spec- 2006). In these cells, the working electrode is a gold grid
troscopy (Weidlich and Siebert 1993) with time resolution (4–7 lm thick) deposited between two visible and infrared
in the ls to ns range (Mezzetti et al. 2003; Kötting and transparent windows (CaF2, or chemical vapor deposition
Gerwert 2005; Sivakumar et al. 2005). In addition, time- (CVD) diamond). In the cells for aqueous samples, the
resolved IR spectroscopy can be achieved using tunable path-length is below 10 lm, the sample volume is less than
infrared laser diodes (Hienerwadel et al. 1990, 1995). For 10 ll, but the concentration of proteins is high, up to the
time resolved techniques, and especially for step-scan millimolar range. An electrochemical cell has also been
techniques, the reaction must remain unchanged for a high developed for ATR (attenuated total reflection)-FTIR
number of repetitive flashes. This restricts the use of this spectroscopy (Rich and Iwaki 2007).
technique to very stable reactions and step-scan techniques
are not easy to apply to photosynthetic reactions centers, Perfusion-induced ATR-FTIR difference spectroscopy
notably PSII.
It is also very important to generate the same reaction- New approaches for FTIR difference spectroscopy involve
induced state in all the active centers of the protein, to the use of ATR, coupled with the perfusion of samples with
obtain reliable FTIR difference spectra. In this respect, the buffers containing a triggering reagent or a metal cofactor
use of alternate spectroscopic controls (UV–Visible, EPR) (see Rich and Iwaki (2007) for a review). With an ATR
is often essential to validate the experimental conditions setup, the infrared beam is reflected within the ATR
(Hienerwadel and Berthomieu 1995; Hienerwadel et al. crystal. At each reflection, the evanescent wave probes a
1996). Thus, FTIR difference spectroscopy is seldom used layer of the sample deposited on the crystal, within a
to identify a new reaction state, but it is very useful to thickness of about 1 lm. In contrast with transmission
analyze the details of the molecular properties of this state. FTIR difference spectroscopy, in which reactions must be
The use of photolabile precursor compounds such as induced in the thin path length sample by external methods,
caged ATP or caged-calcium was implemented to trigger without modifying the sample volume and content, this
light-induced reactions of Ca2?-ATPases or changes in approach has the advantage of minimizing global absorp-
sarcoplasmic reticulum (Barth et al. 1990; Buchet et al. tion changes, when the protein layer deposited on the ATR
1991). The ATP molecule is released from the inactive crystal is exposed to reactants administrated at the top of
precursor by a photolytic UV flash, and then it interacts the sample. This approach was applied to the recording of
with the protein. A number of caged compounds or light- FTIR difference spectra of different redox cofactors of PSI
sensitive molecules are now available, which could be used or bacterial reaction centers dried as stable films on the
for infrared spectroscopy (Barth and Corrie 2002; Mayer ATR crystal (Iwaki et al. 2002). The difference spectra
and Heckel 2006). UV-light excitation was also used to obtained by the perfusion of buffers poised at different

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162 Photosynth Res (2009) 101:157–170

buffer in buffer out assignment of 10a ester and 9 keto C = O groups of the
Valve 1
neutral and cationic forms of bacteriochlorophylls. Simi-
1
Valve 2
larly, the contributions from chlorophyll 9keto and 10a
ester C=O groups in the primary electron donor (P700) of
O-ring Membrane PSI were identified by comparison of the light-induced
2
P700?/P700 spectra with electrochemically induced
(pyro)chlorophyll?/chlorophyll difference spectra in tetra-
3
hydrofurane (Nabedryk et al. 1990a), as well as by using
ZnSe Sample site-directed mutants (Hastings et al. 2001). The frequency
of the (bacterio)chlorophyll IR modes allowed the analysis
IR
of their hydrogen bonding properties and the extent of
Fig. 3 Scheme of the attenuated total reflection (ATR)-microdialysis charge delocalization in primary electron donors of the
setup allowing the perfusion of a protein sample by three different different photosystems (Noguchi et al. 1998; Breton 2001;
buffers Pantelidou et al. 2004).
Similar approaches combining light-induced spectros-
redox potentials were identical to spectra recorded using copy on photosynthetic reaction centers and electrochem-
light-induced FTIR difference spectroscopy (Iwaki et al. ically generated difference spectra on models were used to
2002). This approach was also extended to the analysis of assign the IR contributions of quinones in photosynthetic
soluble proteins maintained on the ATR crystal by a dial- reaction centers (Bauscher et al. 1990; Nabedryk et al.
ysis membrane (Lehmann et al. 2002; Gourion-Arsiquaud 1990b), or to analyze the high and low potential forms of
et al. 2005; see Fig. 3). For this latter application, it is of cytochrome b559 (Berthomieu et al. 1992). A chemically
prime importance to maintain a constant protein concen- generated b-carotene cation in chloroform was used to
tration on the ATR crystal, and to minimize the reaction identify the contribution of a carotenoı̈d cation in light-
time. This was achieved by the setup of a microdialysis induced FTIR difference spectra recorded at low temper-
chamber (Gourion-Arsiquaud et al. 2005; Rich and Iwaki ature in PSII (Noguchi et al. 1994).
2007; Vidaud et al. 2007), which contains less than 5 ll of Absorption spectra of model compounds are also used to
sample. Two or three buffers can be perfused subsequently assign vibrational modes from amino acid side chains in
through the sample, using a peristaltic pump and two three- proteins. This has notably been done to identify IR contri-
way valves, monitored by the FTIR spectrometer. With butions from aspartate, glutamate, tyrosines or histidines and
such a miniaturized system, IR changes corresponding to to identify their hydrogen bonding properties or the role of
single modes are detected (Fig. 3). histidine as metal ligand. IR markers of histidine–metal
With the above-described reaction-induced techniques, interactions have been analyzed notably using 4(5)-meth-
protein–protein interactions, as well as cofactor–protein ylimidazole as a model (Dupeyrat et al. 2004, and references
interactions and protein metal-binding sites can be studied therein). As also observed by resonance Raman spectros-
in a wide range of soluble or membrane proteins. copy (see B. Robert, this issue), the m(C4C5) mode frequency
and its sensitivity to H/2H exchange differ for metal com-
Band assignments plexes in which methylimidazole binds via the Ns or Np
imidazole nitrogens. The ring m(C5 Ns) mode of 4(5)-
Comparison with spectra of model compounds methylimidazole contributing at &1,100 cm-1 is strongly
enhanced upon metal binding (Berthomieu et al. 1992;
Comparison of spectra recorded with protein samples and Dupeyrat et al. 2004). This band, downshifted by &7 cm-1
spectra recorded on model compounds is a usual approach upon 15N labeling, has been observed in a number of proteins
to identify the contributions from cofactors or amino acid and is a useful IR marker of histidine–metal coordination.
side chains in the protein and to discover precise specific
structures or interactions. Theoretical approaches for normal modes calculations
The IR contributions of bacteriochlorophyll cofactors in
the complex P?Q- ?
A /PQA or P /P FTIR difference spectra Experimental data on model compounds are being
recorded with reaction centers from purple bacteria were increasingly analyzed using theoretical approaches to pre-
first analyzed by comparison with electrochemically dict normal mode frequencies. Calculations based on
induced FTIR spectra of neutral and cationic bacterio- density functional methods give satisfactory normal mode
chlorophyll (BChl, BChl?/BChl difference spectra) in predictions. The combined experimental and theoretical
organic solvents of different polarity (Mäntele et al. 1988a, approaches are very powerful in correlating the mode fre-
b; Leonhard and Mäntele 1993). This approach allowed the quencies with specific structures or interactions within the

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Photosynth Res (2009) 101:157–170 163

protein. As an example, the theoretical analysis of the unlabeled

influence of different types of hydrogen bonding interac- 13
C 1(4)-Tyr
tions (proton donor, proton acceptor, or both) on the IR Spectres TyrD °/TyrD

- 1503
(a)
A

- 1468
frequency of tyrosine side-chain modes (Takahashi and
Noguchi 2007) has been useful in interpreting FTIR data
recorded on TyrD in PSII (Hienerwadel et al. 2008; also see
below). Similarly, ab initio normal mode predictions were

1220 -
1250 -
used to better understand the modulation of the m(CO) IR
mode frequency of tyrosinyl radicals as a function of

- 1512
hydrogen bonding interactions (O’Malley 2002). Other Spectres TyrZ°/TyrZ

- 1476
examples concern the normal mode analysis of 4(5)- (b)
B
methylimidazole, a model of the histidine side chain, in
different protonation states or as metal ligand (Hasegawa

-1226 -
et al. 2000, 2002, and references therein) or the analysis of

1255
the hydrogen bonding status of protonated side chains from
aspartate and glutamate (Nie et al. 2005).

Isotope labeling
1700 1600 1500 1400 1300 1200 1100
-1
Wavenumber (cm )

-
Studies on model compounds of both cofactors and amino
acid side chains often imply the use of isotope labeling. Fig. 4 Light-induced FTIR difference spectra corresponding to the
Isotope labeling not only enables band assignments but oxidation of redox-active TyrD (a) and TyrZ (b) of Photosystem II
also helps to determine specific normal modes of vibration, (Hienerwadel et al. 1997; Berthomieu et al. 1998a, b). Spectra were
recorded using PSII core complexes of Synechocystis sp. PCC 6803
which contain structural information. The same labeling with unlabeled tyrosines (thin lines) or tyrosines 13C labeled at the
experiments can be performed on models and in the protein ring carbon involved in the C–O bond (thick lines)
sample, either by substitution of the cofactor (quinones) or
by introducing a labeled precursor in the culture media QB and Q- B , the use of isotopically labeled quinones
for biosynthetic incorporation (hemes or chlorophylls). showed that the two quinone oxygen atoms are involved in
Labeling of the protein itself is obtained by growing the moderate hydrogen bonds (Breton et al. 1995; Brudler et al.
bacteria in a synthetic medium containing either a labeled 1995). Thus, in addition to a precise assignment of quinone
carbon or nitrogen source (global 13C or 15N labelling) or vibrations, detailed structural analysis could be made,
the labelled amino acid (e.g., tyrosine or histidine). Isotope concerning the hydrogen bonding pattern at the carbonyl
edited FTIR spectroscopy has been increasingly used not groups and also concerning steric constraints exerted by the
only to analyze properties of amino acid side chains but protein on quinone substituents (Breton et al. 1995).
also to probe the structure of proteins with high precision. The specific isotope labeling of amino acids has also
The substitution of cofactors by chemically modified or proven useful for a number of spectroscopic techniques as
isotope labelled cofactors is a powerful means to selec- ESEEM (electron spin echo envelope modulation), ENDOR
tively perturb the FTIR spectrum. As an example, the (electron nuclear double resonance) or FTIR spectroscopy.
primary (QA) and secondary (QB) quinones of the bacterial As an example, this strategy has been used to study the
reaction centers of R. sphaeroides and B. viridis have been vibrational properties of two redox active tyrosines (TyrD
substituted by site-specific isotopically labeled quinones. and TyrZ) of PSII, using reaction centers of Synechocystis sp.
For QA, the 18O labelling on both carbonyl oxygens PCC 6803 with labeled tyrosines (Hienerwadel et al. 1997;
allowed the identification of two very different m(C=O) Noguchi et al. 1997; Berthomieu et al. 1998b). 13C-labeling
frequencies, showing an asymmetric hydrogen bonding at the phenolic C4 carbon atom only led to the shift of one
pattern for the two carbonyl groups in the bacterial RC negative band for reduced TyrD (or TyrZ) and one positive
(Breton et al. 1994a). Site-specific 13C labeling, on either band for oxidized Tyr•D (or Tyr•Z; see Fig. 4). This labeling
one of the two carbonyls, or at each carbon positions in the allowed the unambiguous assignment of these bands to the
quinone ring, allowed the assignment of the strong phenol C–O group of reduced and oxidized tyrosines.
hydrogen bond to the carbonyl group distal to the quinone
isoprenoid chain (Breton et al. 1994b; Brudler et al. 1994). Site-directed mutagenesis
In contrast, for the semiquinone Q- A , similar hydrogen
bonding interactions were proposed for the two CO Protein bands can also be identified by the comparison of
bonds (Breton et al. 1994b; Brudler et al. 1994). For both spectra recorded with wild type and site-directed mutant

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164 Photosynth Res (2009) 101:157–170

proteins. This approach has been often used and some photosystem II. In PSII reaction centers of Synechocystis
examples are illustrated in the following section. Special sp. PCC 6803, the rate of oxidation of TyrD dramatically
care must be taken to differentiate between the direct and increases as a function of pH, with a behavior corre-
indirect effects of the mutation. The IR contributions of the sponding to a pKa of &7.6 (Faller et al. 2001). The origin
native amino acid disappear in the mutant. However, the of the group(s) responsible for this apparent pKa was
mutation can slightly alter the protein structure leading to unknown. The analysis of Tyr•D/TyrD FTIR difference
band shifts or spectral changes in the spectra. The combi- spectra on wild type (WT) PSII and PSII with 13C-labeled
nation of site-directed mutagenesis and isotope labeling is tyrosines at different pHs showed that the pH-induced
also powerful in detailed structural information (see below). change concerns the reduced TyrD-state only, and reduced
TyrD itself (Hienerwadel et al. 2008). However, the fre-
quencies of the TyrD IR modes showed that TyrD does not
Typical information gained using FTIR difference deprotonate at pH higher than 7.6, but forms a stronger
spectroscopy of photosystems hydrogen bond, acting as hydrogen bond donor only. At pH
below 7.5, the IR frequencies observed for TyrD indicate
Conformational changes that it could be involved both as a hydrogen bond donor
and acceptor, as judged by a comparison with calculations
FTIR difference spectroscopy is very sensitive to detect performed on models by Takahashi and Noguchi (2007).
conformational changes in proteins, because of the strong The involvement of TyrD in a strong hydrogen-bonding
IR absorption of peptide carbonyl groups. However, one interaction correlates with the ability to oxidize TyrD rap-
striking information obtained from the first comparison of idly at room temperature. This example illustrates the high
light-induced FTIR difference spectra recorded on bacterial precision that can be obtained using FTIR spectroscopy in
reaction center with redox-induced spectra of isolated the analysis of amino acid side chain properties.
cofactors was that the charge separation induced the
perturbation of only a few protein carbonyl groups Proton transfer coupled to electron transfer: direct
(Mäntele et al. 1988a). This holds true for a large number probe of Asp and Glu protonation state
of light-induced reactions in bacterial reaction centers and
photosystems. In bacterial reaction centers and PSII, the secondary elec-
tron acceptor QB is doubly reduced and protonated to form
Hydrogen bonding interactions of cofactors and redox the quinol QBH2 which leaves the reaction center. These
intermediates reactions involve proton transfers across the protein
towards QB, which are coupled to sequential electron
FTIR spectroscopy is not the only technique which allows transfers from Q- A.
the detection of hydrogen bonding interactions on the FTIR spectroscopy is the method of choice to probe
various cofactors in the reaction centers. ENDOR and directly protonation changes of aspartate and glutamate side
ESEEM also give precise analysis of interactions involving chains, by following absorption changes of the m(C = O)
singles atoms in a molecule. However, these techniques mode of the protonated carboxylic (COOH) groups in the
can only probe radical species, i.e., the oxidized or reduced 1,770–1,700 cm-1 region.5 This approach has been used to
intermediates of the reaction. They do not provide infor- determine the role of Asp and Glu residues located near QB in
mation on the fine structure of the cofactors in the resting proton uptake upon Q- B formation in bacterial reaction cen-
state. We have illustrated above how the use of specific ters (reviewed in Nabedryk and Breton 2008). In the first
isotope labeling of the quinones revealed the hydrogen experiments using time-resolved IR (Hienerwadel et al.
bonding interactions of QA and QB in the dark-adapted 1995; see Fig. 5) and FTIR (Nabedryk et al. 1995) spec-
resting state of the bacterial reaction center. This approach, troscopy on WT and GluL212Gln mutant reaction centers of
coupled with the analysis of FTIR difference spectra Rb. sphaeroides, the authors assigned a positive band at
obtained with site directed mutants, further showed that 1,728 cm-1 to the protonation of a fraction of GluL212 side
only one QB binding site was observed in the FTIR sam- chain upon Q- B formation at pH 7. Indeed, the 1,728 cm
-1
-1 2
ples. These results contrasted with crystallographic studies band was downshifted by &10 cm upon H/ H exchange
that proposed two -distal and proximal- locations for QB. (typical shift for a COOH group exchangeable with the
They showed that all QB molecules involved in electron
transfer from Q- A were located at the proximal binding site 5
In contrast, the identification of the mas and ms (COO-) modes of the
(reviewed in Nabedryk and Breton 2008).
corresponding carboxylate forms is often impaired by many other
Another example is the analysis of hydrogen bonding superimposed IR modes in the 1,610–1,550 and 1,420–1,380 cm-1
interactions formed by reduced TyrD in Mn-depleted region. Isotope labeling is useful in identifying these modes.

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Photosynth Res (2009) 101:157–170 165

(a)
native protein
∆Α
0.1*10 -3
(a)

mutant Glu L212 GLN

1ms

(b) 1339
1530
1600 1205
(b)
relative amplitude

1658 1495
1230
1305

1700 1600 1500 1400 1300 1200 1100

Wavenumber (1/cm)

OH
(c)
C
QB
O O H
2+ N

N
N
N
Fe N
H
D2–His215 N D1–His215 OH
QA
HN C
O
Fig. 5 a Time resolved IR changes recorded at 1725 cm-1 with D2–His269 NH
O
D1–His272
reaction centers from Rb. sphaeroides WT (wild type, native protein) N
N Fe3+ N
and GluL212Gln mutant (Hienerwadel et al. 1995). b IR amplitude of N N
N
the slow phase component corresponding to the proton uptake as a QA
HN
function of pH NH

Fig. 6 a Light-induced Fe2?/Fe3? FTIR difference spectra recorded

solvent) and disappeared in the GluL212Gln mutant (Fig. 5). with Photosystem II enriched membranes of spinach in the presence
The time resolved study also showed that the proton uptake of 12C-bicarbonate (thin line) or 13C-labeled bicarbonate (thick line).
by GluL212 occurred in a broad pH range (pH 6–10). These b 12C-minus-13C difference spectrum calculated from the spectra in
results were in disagreement with the interpretation of pH A; c structural changes upon Fe oxidation resulting from the
interpretation of the FTIR data
dependence of electron transfer rate or proton uptake
experiments on WT and mutants that suggested proton identify amino acid metal ligands and to analyze the
uptake by GluL212 at high pH only. However, the proton properties of metal–ligand interactions. This is illustrated
transfer mechanisms associated with Q- B formation is a by the detailed analysis of the correlation between the
typical example, where the interpretation of global effects is mode of binding of carboxylate groups to metals and the
complicated by strong interactions between several groups. frequency of the asymmetric (mas) and symmetric (ms)
In IR spectroscopy, each residue has its own and single stretching mode frequencies (Deacon and Phillips 1980;
signature. Therefore, this technique is particularly suited to Nakamoto 1997).
analyze such complex situations (Nabedryk and Breton This property was used to propose that bicarbonate is a
2008). Further experiments on a large number of mutants bidentate ligand of the non heme iron of PSII in the Fe2?
confirmed that GluL212 was responsible for the IR band at state (see Fig. 6; Hienerwadel and Berthomieu 1995). In
1,728 cm-1, and that it was the only carboxylic group getting this study, light-induced Fe2?/Fe3? FTIR difference spec-
partially protonated upon Q- B formation at pH 4–11 (Nabe- tra recorded with samples containing unlabeled or 13C-
dryk and Breton 2008). labeled bicarbonate were compared to allow an unambig-
uous assignment of the bicarbonate IR modes. Indeed, the
Identification and properties of metal ligands superimposition of the two spectra (Fig. 6a) shows that
only a few IR bands appear at different frequencies in these
The sensitivity of vibrational modes of amino acid side two spectra. These bands are bicarbonate IR modes both
chain groups to interactions with metals can be used to sensitive to bicarbonate 13C-labeling and to the change in

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166 Photosynth Res (2009) 101:157–170

redox state of the non-heme iron. The assignment of these We have previously shown that the IR modes of histi-
IR bands, extracted in the 12C-minus-13C difference spec- dine can also be used to identify its role as a metal ligand.
trum (Fig. 6b), was done considering that a downshift of Signals at 1,102 and 1,094 cm-1, sensitive to 15N-labeling,
the bicarbonate IR modes is expected upon bicarbonate were detected in the Fe3? state of the non-heme iron of
13
C-labeling. The two bands at 1,530 and 1,338 cm-1 were PSII (Fig. 6a; Hienerwadel and Berthomieu 1995) and
assigned to the mas and ms (COO-) modes of bicarbonate in therefore assigned to histidine ligands of the iron. The
the Fe2? state. The frequency difference of 192 cm-1 is band at 1,094 cm-1 was perturbed upon o-phenanthroline
lower than that observed for bicarbonate in solution binding at the QB binding site, strongly suggesting that it is
(&285 cm-1), leading to the conclusion that bicarbonate is due to D1-His215 (Fig. 6c; Berthomieu and Hienerwadel
a bidentate ligand of Fe2? (see Hienerwadel and Bertho- 2001). The frequency of the His 1,094 cm-1 band was
mieu 1995 for the details in assignment). In the Fe3?-state, taken as indicative of deprotonation of the histidine side
the bicarbonate mas and ms (COO-) modes were identified at chain upon iron oxidation, as illustrated by the reaction
1,658 and 1,230 cm-1. These frequencies indicate that model proposed in Fig. 6c.
bicarbonate is a monodentate ligand of Fe3? (Fig. 6b). IR contributions at &1,114 cm-1 in the spectra of
These data support the extensive work of Govindjee and S-state transitions, sensitive to 15N-labeling, were also
coworkers that show that bicarbonate plays an essential assigned to one histidine ligand of Mn, possibly D1His332
role in the electron and proton transport in the QA QB (Noguchi et al. 1999; Yamanari et al. 2004; Kimura et al.
region of PSII (see e.g., Van Rensen et al. 1999; Rose et al. 2005). This histidine is observed in the S0–S1, S1–S2 and
2008). S2–S3 S-states transitions. The effect of deuteration on that
For PSII, one main challenge is the elucidation of the mode was taken as indirect evidence that this histidine
molecular mechanism of water oxidation at the Mn4Ca ligates the Mn by the imidazole Ns nitrogen (Noguchi et al.
active site (see Wydrzynski and Satoh 2005). In this respect, 1999).
FTIR difference spectroscopy plays a central role in iden-
tifying the Mn–ligands and the changes in ligand–Mn Properties and role of water molecules
(ligand–Ca) interactions during the sequential steps leading
to water oxidation. This is challenging in the context where A particularly attractive possibility of FTIR spectroscopy is
the three dimensional X-ray structures most probably do not the direct analysis of internal water molecules with active
correspond to physiological oxidation states of the Mn roles in proton transfer or in catalysis in proteins. Such
cluster. Details of the results obtained using FTIR difference water molecules are present in bacteriorhodopsin, bacterial
spectroscopy of the oxygen evolving complex are given in reaction centers, and in PSII. For the latter system, the
several reviews (Noguchi and Berthomieu 2005; Noguchi direct probing of substrate water during water oxidation at
2007; Debus 2008,). In particular, the use of universal 13C the oxygen evolving center is appealing.
and 15N labeling allowed the identification of a number of The water molecules can be directly probed by their
carboxylate groups: the mas and ms (COO-) modes were m(OH) modes in reaction-induced FTIR difference spectra.
identified by their large sensitivity to 13C-labeling (25– This is, however, challenging due to the large background
50 cm-1 downshifts) and their insensitivity to 15N labeling absorption of the water in the sample suspension. The study
(Noguchi and Sugiura 2003; Yamanari et al. 2004). Com- of water molecules has been undertaken at low tempera-
parison of spectra recorded with native PSII and PSII tures (Yamazaki et al. 1995; Kandori 2000; Noguchi and
depleted of calcium further showed the disappearance of Sugiura 2000) or on partially dehydrated protein films at
band pairs at 1,587 cm-1 (S2-state)/1,560 cm-1 (S1-state) the room temperature (Noguchi and Sugiura 2002; Lorenz-
and 1,364 cm-1 (S2-state)/1,403 cm-1 (S1-state), which Fonfria et al. 2008).
were assigned to the mas and ms (COO-) modes of a single The broad absorption of water or ice prevents analysis of
carboxylate group. As discussed above for bicarbonate, the the 3,500–3,000 cm-1 range. However, at low tempera-
mas - ms frequency differences of 157 cm-1 in the S1 state tures, the sample absorption is very stable and the
and of 223 cm-1 in the S2 state were taken as indicative of a difference bands flanking this region are detected in light-
drastic change in coordination of this carboxylate—from induced FTIR difference spectra. At the room temperature,
bridging to unidentate coordination—to a Mn of the oxygen partial dehydration of the protein sample is controlled by
evolving complex (Noguchi et al. 1995). The assignment of the addition of droplets of water/glycerol mixtures in
each carbonyl group to a specific amino acid involves now proximity of the protein film, placed in a tight measure-
the use of site directed mutants sometimes in combination ment cell (Noguchi and Sugiura 2002a). The water/glycerol
with specific isotope labeling (Chu et al. 2004; Strickler ratio determines the humidity of the cell and hence of the
et al. 2006, 2007; see reviews by Debus 2008 and Noguchi protein film. Using this approach, Noguchi and Sugiura
2008). (2002b) have identified contributions from water molecules

123
Photosynth Res (2009) 101:157–170 167

during the S-state transitions of the oxygen evolving Therefore, although this technique requires well defined
complex of PSII from Synechococcus elongatus. The experimental conditions and the use of site directed mutants
assignment of the water bands resides on their sensitivity to or isotope labeling, it will continue to play a key role in the
H2O/H18 2 O exchange. Bands observed at 3,618 cm
-1
(S2- analysis of photosystems and other proteins, and notably to
-1
state) and 3,585 cm (S1-state) were thus assigned to a unravel the mechanism of water oxidation in PSII.
water molecule sensitive to Mn oxidation during the S1 to The remarkable increase in the number of publications
S2 transition of the water oxidizing complex. on proteins involving (FT)IR spectroscopy shows the
The m(OH) mode frequency strongly depends on important and specific role of this technique. The contin-
hydrogen bonding interactions. Moreover, for water mol- uous development of new and complementary experimen-
ecules involved in symmetric interactions, the two m(OH) tal strategies and theoretical approaches opens its field of
vibrations are coupled, resulting in an asymmetric and in a application in various research areas.
symmetric mode. In contrast, water molecules involved in
asymmetric interactions have two uncoupled m(OH) modes Acknowledgment This manuscript was edited by Govindjee.
at different frequencies. To measure the extent of intra-
molecular coupling of the water molecules, samples in H2O
are compared with samples in 50% of H2O and 50% 2H2O: References
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