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

Isotope-edited infrared spectroscopy
Metal–ligands

Water molecules
List of abbreviations
ATR Attenuated total reflection
BChl Bacteriochlorophyll
CVD Chemical vapor deposition
ENDOR Electron nuclear double resonance
ESEEM Electron spin echo envelope modulation
FTIR Fourier transform infrared
IR Infrared
P
700
Primary electron donor of PSI
PSI Photosystem I
PSII Photosystem II
Q
A
,Q
B
Primary and secondary electron acceptor
quinones of photosynthetic RCs
RC Reaction center
Tyr
D
,Tyr
Z
The two redox active tyrosines of PSII
WT Wild type
Introduction
Infrared (IR) or Fourier transform infrared (FTIR) spec-
troscopy has a large application range, from the analysis of
Special Issue of Photosynthesis Research ‘‘Basics and Applications of
BiophysicalTechniques in Photosynthesis and Related Processes’’,
edited by Messinger, Alia and Govindjee.
C. Berthomieu (&)
CEA (Commissariat a` l’ Energie Atomique), Laboratoire des
Interactions Prote´ine Me´tal, DSV (Direction des Sciences du
Vivant), Institut de Biologie Environnementale et
Biotechnologie, Service de Biologie Ve´ge´tale et Microbiologie
Environnementales (DSV/iBEB/SBVME), CEA-Cadarache,
UMR 6191 Centre National de la Recherche Scientifique
(CNRS)-CEA (Commissariat a` l’ Energie Atomique)-Universite´
Aix-Marseille II, 13108 Saint Paul-lez-Durance Cedex, France
e-mail: catherine.berthomieu@cea.fr
R. Hienerwadel
Aix-Marseille Universite´, Laboratoire de Ge´ne´tique et de
Biophysique des Plantes, Faculte´ des Sciences de Luminy,
Direction des Sciences du Vivant, Institut de Biologie
Environnementale et Biotechnologie, Service de Biologie
Ve´ge´tale et Microbiologie Environnementales (DSV/iBEB/
SBVME), UMR 6191 Centre National de la Recherche
Scientifique (CNRS)-CEA (Commissariat a` l’ Energie
Atomique)-Universite´ Aix-Marseille II, 13288 Marseille
cedex 9, France
123
Photosynth Res (2009) 101:157–170
DOI 10.1007/s11120-009-9439-x

small molecules or molecular complexes to the analysis of
cells or tissues. The imaging of tissues is one of the recent
developments of infrared spectroscopy, taking advantage
of infrared microscopy and of the use of synchrotron IR
radiation. It is used for the mapping of cellular components
(carbohydrates, lipids, proteins) to identify abnormal cells
(Levin and Bhargava 2005; Petibois and De´le´ris 2006).
FTIR spectroscopy has also been increasingly applied to
the study of proteins. This concerns the analysis of protein
conformation, protein folding, and of molecular details
from protein active sites during enzyme reactions using
reaction-induced FTIR difference spectroscopy (Siebert
and Hildebrandt 2008).
FTIR difference spectroscopy has been widely applied
in photosynthesis research and related areas. This approach
gives complementary information to the three-dimensional
structural data obtained by X-ray diffraction (or Nuclear
Magnetic Resonance, NMR). The analysis of active sites in
proteins by means of reaction-induced FTIR difference
spectroscopy gives information on minute structural
changes, hydrogen-bonding interactions, and proton trans-
fer reactions, which are often beyond the sensitivity of X-
ray diffraction analyses. Moreover, time-resolved tech-
niques, with time resolution now up to the femtosecond
range (Di Donato et al. 2008) allow structural changes to
be observed for protein active sites ‘‘at work’’.
In photosynthesis, this approach has given information
of prime interest concerning the cofactor-protein interac-
tions (Nabedryk 1996; Breton 2001; Berthomieu and
Hienerwadel 2005; Noguchi and Berthomieu 2005), as well
as proton transfer routes in bacterial reaction centers
(Nabedryk and Breton 2008). It now plays a central role in
the detailed analysis of the oxygen evolving complex of
Photosystem II (Chu et al. 2001; Noguchi 2007; Debus
2008).
While this approach suffers from several limitations, it
delivers unique information by addressing directly the
properties of cofactors, amino acids, and water molecules,
with very high sensitivity to structural parameters and
electronic interactions. This justifies the experimental
efforts that have been made to optimize its use and the
interpretation of the data.
In the following, we briefly present the principles of
infrared spectroscopy and describe the development of
experimental approaches to identify and analyze IR sig-
natures from active sites in proteins by reaction-induced
FTIR difference spectroscopy. We describe the methodol-
ogy to obtain reliable spectra and interpretations, and show
typical examples of specific information brought by this
technique in the study of photosystems.
1
Principle and methodology of infrared spectroscopy
and FTIR difference spectroscopy
Infrared spectroscopy
Infrared spectroscopy probes the molecular vibrations.
Functional groups can be associated with characteristic
infrared absorption bands, which correspond to the funda-
mental vibrations of the functional groups (Colthup et al.
1975; Griffith and de Haseth 1986). For a nonlinear mol-
ecule with N atoms, there are 3N-6 vibrational motions of
the molecule atoms, or 3N-6 fundamental vibrations or
normal modes. A normal mode of vibration is infrared
active (i.e., it absorbs the incident infrared light) if there is
a change in the dipole moment
2
of the molecule during the
course of the vibration. Thus, symmetric vibrations are
usually not detected in infrared. In particular, when a
molecule has a center of symmetry, all vibrations which are
symmetrical with respect to the center are infrared inactive.
In contrast, the asymmetric vibrations of all molecules are
detected. This lack of selectivity allows us to probe the
properties of almost all chemical groups in one sample, and
notably of amino acids and water molecules which can
hardly be observed by other spectroscopic techniques.
Strong IR absorptions are observed for groups with a
permanent dipole (i.e., for polar bonds). As such, the car-
bonyl groups of the polypeptide backbone contribute lar-
gely to the infrared absorption spectra of proteins.
In the mid-infrared region (4,000–1,000 cm
-1
), two
main types of vibrations are observed: vibrations along
chemical bonds, called stretching vibrations (m), which
involve bond-length changes; and vibrations involving
changes in bond angles, and notably bending vibrations
(d—in plane, p—out of plane).
The stretching vibrations can be modeled using the
harmonic oscillator model (Fig. 1), in which a chemical
bond is represented by two point masses linked by a spring.
The bond strength (or molecular force field) is the spring
tenseness k and the point masses (m
1
and m
2
) model the
masses of the atoms or chemical groups involved in the
bond. The oscillation frequency m is given by the equation
3
:
m ¼ 1=2pcðÞ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
k m
1
þ m
2
ðÞ=m
1
m
2
Þ
p
:
The vibration frequency m thus depends on the bond
strength, with higher frequencies for triple or double bonds
as compared to single bonds. This is illustrated in Fig. 1
for CO bonds. A consequence of the dependence of
stretching mode frequencies on the bond strength is that the
1
We do not present an exhaustive review of the FTIR literature on
photosystems.
2
Dipole moment: when a positive charge ?z and a negative charge
-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). .
3
In the FTIR spectra, the infrared absorption is given as a function of
the vibration frequency expressed in cm
-1
.
158 Photosynth Res (2009) 101:157–170
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