Fluorescence-emission spectroscopy of individual LH2 and LH3 complexes
Introduction
The first step in photosynthesis is the absorption of photons by pigment–protein complexes that are specialized in light harvesting (LH). They efficiently transfer the excitation energy to a reaction center (RC), where charge separation takes place [1]. Collective properties of the pigment molecules play an important role in the excitation dynamics and spectral properties of such systems, which are usually described in terms of exciton models. In cases where the structure of the pigment–protein complexes is available from X-ray crystallography, model calculations and predictions can be based on detailed structural information, which can be compared with data from experimental studies of the intrinsic photo-physical properties of light harvesting complexes.
Optical spectroscopy of these systems at the level of individual complexes provides direct insight into their electronic properties, avoiding the inhomogeneous broadening that is typical for ensemble spectra, while providing easy access to disorder parameters [2], [3], [4], [5], [6], [7], [8], [9]. In previous publications we reported the optical absorption spectra of individual light harvesting complexes LH2, LH3 and LH4, respectively, from Rhodopseudomonas (Rps.) acidophila and Rps. palustris [6], [7], [8]. Here we address the fluorescence-emission spectra of single LH2 and LH3 complexes. In these systems the fluorescent state differs from the states that determine the absorption spectra. Therefore, the emission spectra provide information that is not readily accessible in fluorescence detected absorption spectra. The emitting state of single complexes is furthermore an excellent reporter on the stability of the protein and its local environment [9].
LH3 is a spectroscopic variant of the extensively studied LH2 complex. The structures of LH3 and LH2 have both been resolved at high resolution by X-ray crystallography [10], [11]. They show that the overall structure of the two complexes is very similar. In fact, their pigment arrangements are practically indistinguishable. Both LH3 and LH2 have a nine-fold circular repetition of subunits consisting of an αβ-protein pair, each containing three bacteriochlorophyll a (BChl a) molecules and one carotenoid molecule. The 27 BChl a molecules in the complexes are arranged in two concentric rings, the B800 and B820 rings in the case of LH3 and the B800 and B850 rings in LH2. The nomenclature for the rings refers to the spectral position of their respective absorption bands [12]. The band absorbing at 800 nm consists of nine monomeric BChl a pigments with their macrocyles in the membrane plane, while the remaining 18 BChl a pigments in the B820 band of LH3 are oriented perpendicular to the membrane plane, like the B850 BChls in LH2. The pigments in the latter ring are strongly coupled due to the near anti-parallel alignment of their dipole moments and the short distances (about 9 Å center-to-center) between adjacent pigments.
In this paper, we present the results of fluorescence-emission spectroscopy on individual LH2 and LH3 complexes at low temperature. Downward relaxation in the exciton manifold of light harvesting pigment–protein complexes typically occurs on a sub-picosecond time scale [13], [14], [15]. Under the low temperature conditions of our measurements, this implies that fluorescence originates from the lowest state in the exciton manifold, whereas most of the oscillator strength is contained in higher exciton states.
Our data reveal large differences between the spectra of the two LH complexes. We attribute these differences to spectral diffusion processes that are characteristic for each of the complexes. Also the summations of the single-complex spectra display distinct behavior. We elaborate on the structural differences between the complexes that may contribute to the observed effects. Numerical calculations based on the structural models of both complexes are used to analyze the results in more detail. We have analyzed the fluorescence properties of LH2 and LH3 at the single-molecule level to further extend the exciton model, and to investigate effects of static and dynamic heterogeneity.
Section snippets
Materials and methods
Isolation and purification of LH2 (strain 10050) and LH3 (strain 7050) from Rps. acidophila was performed as described previously [16], [17]. Samples for single-complex studies were prepared as in earlier work from our group [7], [8], [18]. Briefly, the stock solution was utilized for bulk as well as single-complex experiments. For the single-complex experiments, the stock solution was diluted with a buffer containing 20 mM Tris, 0.1% LDAO and 1.8% PVA at a pH of 8. The concentration of the
Results
Fluorescence-emission spectra of individual LH2 and LH3 complexes were measured at low temperature (1.2 K) and typical spectra are displayed in Fig. 1. A detailed analysis of the fluorescence quantum yield of LH3 is not available, but under comparable excitation conditions the fluorescence intensity of individual complexes (typically around 100 photon counts per second maximum at an excitation intensity of 100 W/cm2) is rather similar for LH2 and LH3. The majority of the LH2 (Fig. 1a) and LH3
Discussion
The fluorescence spectra of LH2 and LH3 have allowed us to examine the properties of the k = 0 exciton state of individual complexes. The most striking feature of the current results is the large FWHM of the bands in the LH2 and LH3 spectra (Fig. 2). The shape of the fluorescence bands is presumably governed by a superposition of a very narrow zero-phonon line (ZPL) and a broad phonon side-band (PSB). The average width of the experimental spectra, however, is much broader than that of a
Conclusions
We have reported on single-molecule fluorescence-emission experiments comparing LH2 and LH3 and have performed elementary model-based simulations. We have focused our attention on the widths of the individual spectra and on their summation. The widths of the LH2 spectra are much broader than the lifetime limit, and the LH3 spectra are even twice as broad as the LH2 spectra. This is attributed to light-induced spectral diffusion, which is much more pronounced in the case of LH3.
For both, LH2 and
Acknowledgements
On the occasion of this special issue, T.J.A. wishes to acknowledge the role of Prof. Douwe A. Wiersma as a very inspirational and great mentor in the early stages of his carreer.
This work is supported by the Stichting voor Fundamenteel Onderzoek der Materie (FOM) and the Section Earth and Life Sciences (ALW) of the Netherlands Organization for Scientific Research (NWO). S.O. acknowledges financial support from the EU Marie Curie programme for this work. R.J.C. thanks the BBSRC for financial
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Single-molecule spectroscopy reveals that individual low-light LH2 complexes from Rhodopseudomonas palustris 2.1.6. Have a heterogeneous polypeptide composition
2009, Biophysical JournalCitation Excerpt :In other words, some may have site energies characteristic of B850 and some may have ones characteristic of B820. Therefore, the simulation was extended to introduce two different site energies—E0 (αB850) = 12,300 cm−1, E0 (βB850) = 12,060 cm−1 (B850-pair) (42); and E0 (αB820) = 12,860 cm−1, E0 (βB820) = 12,600 cm−1) (B820-pair) (44)—for the Bchl a-pairs in which the B850- and B820-pairs of Bchl a are randomly distributed in the ring (Fig. 4, C and F). In this case the 9-mer model shows the gain of the oscillator strength of all of the exciton states (Fig. 4C).
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2013, Angewandte Chemie - International Edition
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Present address: Laboratory of Physical Chemistry of Polymers and Membranes, ISIC, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
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Present address: Experimental Physics IV, University of Bayreuth, 95440 Bayreuth, Germany.