Broadband 2 D Electronic Spectroscopy Reveals Coupling Between Dark 1 Bu-State of Carotenoid and Qx State of Bacteriochlorophyll

The study of LH2 protein of purple bacteria by broadband 2D electronic spectroscopy is presented. The dark 1Bu carotenoid state is directly observed in 2D spectra and its role in carotenoid-bacteriochlorophyll interaction is discussed.


Introduction
Chlorophylls and carotenoids are the main light absorbing pigments in light-harvesting proteins.Carotenoids have two different functions.They harvest the light energy and transfer it to chlorophylls.They also accept excess excitation energy from chlorophylls and dissipate it, thereby preventing formation of singlet oxygen which can damage the protein.
In the LH2 complexes of Rps.acidophila the light energy, absorbed by lowest allowed S 2 excited state of carotenoid (Car), either relaxes to dark Car S 1 state or is transferred to Q x state of bacteriochlorophyll (BChl).The overall efficiency of energy transfer from Car S 2 state to BChl Q x state is between 40-60% [1][2][3][4].Theoretical study however predicted only 20% efficiency of Car-to-BChl transfer due to small spectral overlap [5].Thus, the energy transfer between the two molecules is substantially contributes to the overall light-harvesting process.However despite active studies, no clear understanding of the underlying processes and mechanisms is present.The experimental obstacle is the strong overlap of the Car/BChl signals both spectrally and temporally.In several works on isolated carotenoids and light-harvesting proteins a signature of additional Car dark state (1B u -or S x state) was found, which increases the controversy of the interpretation of spectroscopic data [4,[6][7][8].
In the current study the 2D electronic spectroscopy was applied to the LH2 complexes of Rps.acidophila.The 2D spectroscopy gives more information than traditional transient absorption, providing spectral resolution along both excitation and emission energy scales.Broadband pulses in the spectral region of 500-630 nm were used in order to excite simultaneously both Car S 2 state and BChl Q x state.The resulted 2D spectra revealed in addition to signals from Car S 2 state and BChl Q x state, a contribution from previously unobserved intermediate state.The origin of this state and its function in LH2 protein is discussed.

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Materials an
LH2 complexe ribed [9].The cell was used ude photo-degr The 2DES setu tfire, Spectra-P e-built NOPA HM).The com nged in the bo mrock, Andor) e corresponds excitation freq a time step of ys (waiting tim  ) is observed at 560 nm wavelength.The decay sampled at the 1B u -peak (Figure 1C green dots) shows very similar oscillations to the oscillations observed at the S 2 peak (Figure 1C blue dots).These decays can be fitted with a sum of exponentials and three frequency modes: 1000 cm -1 , 1250 cm -1 and 1600 cm -1 (see red line in Figure 1C), known to be a signature vibrational frequencies of carotenoids [11].Since the S 2 state is not excited in Figure 1B, the 1B u -peak is due to an additional carotenoid state, which has features very similar to the features of the dark 1B u -(or S x ) state, predicted theoretically [12] and extensively discussed in the literature (for review see [13]).As follows from the 2D spectrum in Figure 1B, Car 1B u -state strongly interacts with the BChl Q x state.The Car 1B u -/BChl Q x cross peak indicates substantial 1B u -→Q x energy transfer.At the same time no Car S 1 ESA is observed after excitation of the 1B u -state, indicating no 1B u -→Q x energy transfer.The summarised electronic level scheme and observed energy pathways are shown in Figure 1D.Appearance of the dark 1B u -is ascribed to the borrowing of the dipole moment strength from the BChl Q x state.Presence of 1B u -→Q x energy transfer can explain the disagreement between experiment and theory in the energy transfer efficiency since the Car 1B u -state was not accounted for in the theoretical studies [1][2][3][4][5].

Conclusions
In this work we report 2D spectra with clear presence of the Car 1B u -dark state.This state has been controversially discussed both in experimental and theoretical studies for the last decade, however no direct observation (as a ground state bleach) of that state in the spectrum has been reported before.Due to the very low dipole moment the Car 1B u -state is invisible the stationary spectra and can only be observed under selective excitation conditions.The substantial contribution of the 1B u -state to the interaction between carotenoid and bacteriochlorophyll molecules is demonstrated.Taking into account the Car 1B u -state is essential for understanding of the energy dynamics in lightharvesting proteins.