**Time-Resolved FTIR Difference Spectroscopy Reveals the Structure and Dynamics of Carotenoid and Chlorophyll Triplets in Photosynthetic Light-Harvesting Complexes**

Alexandre Maxime and Rienk van Grondelle *VU University of Amsterdam Netherlands* 

## **1. Introduction**

230 Infrared Spectroscopy – Life and Biomedical Sciences

Xiaobo, Z., Jiewen, Z. & Yanxiao, L. (2006). "Selection of the efficient wavelength regions in

FiPLS models." *Vibrational Spectroscopy*: 1-8.

FT-NIR spectroscopy for determination of SSC of 'Fuji' apple based on BiPLS and

Infrared spectroscopy is a very powerful tool to determine the chemical nature of molecules.

Differential infrared spectroscopy allows to select only those chemical vibrations involved in a light induced reaction. The structure and environment of unstable and short lived excited states can be probed by using step scan FTIR time resolved spectroscopy. In this chapter we present an application of time resolved FTIR step scan spectroscopy to the light harvesting complexes involved in the collection of solar energy in photosynthesis. The time resolved data are analysed using a global and target analysis procedure which allows identification of the dynamic and the spectral properties of short lived intermediates such as triplet states. Triplet state of chlorophyll a (Chl a) can react with oxygen and lead to the formation of singlet oxygen. Carotenoids avoid this reaction via triplet excitation energy transfer (TEET) and quench the triplet of Chl a. The peridinin chlorophyll protein (PCP), an algal light harvesting complex, which binds Per and Chl a is a good system to study photoprotection mechanism by infrared spectroscopy. Indeed Per and Chl a have both conjugated carbonyl groups that are efficient probes of the molecular state in the infrared. We first investigated by step scan spectroscopy the TEET reaction of Per and Chl a in solvent to get their respective spectral signature. Such a study leads to the identification of several mechanisms associated with the formation of triplet states in solution. Secondly the triplet formation is observed in two different PCP complexes leading to the unexpected conclusion that the Per triplet state is delocalised over the Chl a. In a third part we reveal that the same process of triplet sharing between Chl and carotenoid is also present in higher plants, in sharp contrast with purple bacteria for which the triplet is fully localised on the carotenoid. Our finding strongly suggests that in higher plants and algae a much stronger interaction between carotenoids and chlorophylls is at the basis of photoprotection, and represents an example of molecular adaptation in oxygenic photosynthesis.

Time-Resolved FTIR Difference Spectroscopy Reveals the Structure and Dynamics

interferogram.

with ( ) *<sup>i</sup> k t <sup>i</sup> c e*

of Carotenoid and Chlorophyll Triplets in Photosynthetic Light-Harvesting Complexes 233

ultrafast mid-IR which uses a spectrograph containing a grating to disperse the probe beam on a 32-element MCT detector, Fourier-Transform Infrared (FTIR) spectroscopy is an interferometric method. The FTIR spectrometer consists of an infrared source, an interferometer, the sample, and the infrared detector. The interferometer is the heart of the spectrometer and consists in its simplest form of a beam splitter, a fixed mirror, and a moving mirror scanning back and forth. Therefore, the spectrum is not directly measured but its interferogram, i.e. the IR intensity reaching the detector as a function of the mirror position. The spectrum is subsequently obtained by Fourier transformation of the interferogram. The major advantages of FTIR spectroscopy, as compared to conventional dispersive IR spectroscopy, are the so-called multiplexing advantage (Felgett advantage) and the high energy flux reaching the detector (Jacquinot advantage), allowing rapid spectrum acquisition at a high signal to noise ratio. In the step-scan mode, the interferometer moving mirror may be visualized as being held stationary at the interferogram mirror position xn. The protein activity is initiated, by a laser flash, and the time dependence of the intensity change at this interferogram position xn is measured. Then the interferometer "steps" to the next interferogram data position xn+1, and the reaction is repeated and measured again. This process is continued at each sampling position of the

**2.3 Data analysis for time-resolved infrared spectra. Global and target analysis** 

signal S at any given time (t) or wavenumber (υ) can be described with:

TrIR experiments result in a 3-dimensional dataset as the changes in intensity (*ΔOD*(υ,t)) are measured as a function of time and wavelength. A typical dataset results in ~20 000 data points. To analyze such large amounts of data a Global analysis procedure is required (van Stokkum, Larsen et al. 2004). Global analysis implies a simultaneous analysis of the entire 3 dimensional dataset which gives a correlation between different wavelength-regions and time scales. The total dataset is a superposition of contributions from different species (components) having their own time constants (lifetime). Description of such a total dataset should be obtained with a minimum amount (i) of time constants (ki) that result in a good quality fit. Each component starts with a given concentration (ci) that decays in time and possesses its own specific time-independent spectrum (εi). Mathematically, the observed

> 1 ( , ) ( )\* ( ) *n*

The resulting spectra εi may represent mixture of known physico-chemical species and then do not contain directly physically relevant information. In such case decisions have to be made about a model that not only describes the raw data, but also generates relevant and meaningful spectra. Data previously obtained using different experimental techniques can be helpful in such context. The simplest model describing the measured data has to be chosen. The simplest model templates are the sequential and parallel models (Figures 1A and 1B) where one compartment flows directly into the next compartment with increasing lifetimes or to the ground state, respectively. A compartment represents a spectroscopic distinct state or physico-chemical species and symbolizes a component of the reaction with

*i S t ct* 

*i i*

 

(2)
