Rienk van Grondelle

Spectroscopic properties of LHC-II, the main light-harvesting chlorophyll a/b protein complex from chloroplast membranes

The major chlorophyll a/b light-harvesting complex LHC-II was isolated from spinach thylakoid membranes after solubilization with the non-ionic detergent n -dodecyl- β - d -maltoside. The complex is characterized by a chlorophyll a to b ratio of 1.5±0.1. The spectroscopic properties of this trimeric complex were analyzed and found to be very similar to those observed in the intact thylakoid membrane. By low-temperature absorption, linear dichroism and circular dichroism spectroscopy it is shown that the chlorophyll Q y(0−0) absorption region of the complex is characterized by at least six and perhaps nine chlorophyll transitions with significant differences in energy, orientation and rotational strength. Excitation of the pigments resulted in a sharp fluorescence band peaking near 680 nm at 77K. Presumably, this fluorescence originates from the main and red-most chlorophyll a Q y transition, which peaks at 676 nm and is oriented at a small angle with the plane of the particle. Fitting of polarized excitation spectra indicated that the polarization of Chl 676 is about 0.1; this argues against a single emitting species, but is in line with a model in which efficient energy transfer occurs among several (>3)Chl 676 molecules which form a circularly degenerate oscillator in a plane. Since the shapes of the linear dichroism and polarized excitation spectra were found to be virtually identical between 640 and 676 nm, this plane is probably the same as the plane of the particle. Above 676 nm, the polarization rises gradually to a value of about 0.2, which is interpreted as a result of inhomogeneous line broadening of the 676 nm transition. A model that describes the chlorophyll organization of LHC-II predominantly in terms of exciton interactions between several chlorophyll a and several chlorophyll b molecules is discussed.

Controlled Disorder in Plant Light-Harvesting Complex II Explains Its Photoprotective Role

The light-harvesting antenna of photosystem II (PSII) has the ability to switch rapidly between a state of efficient light use and one in which excess excitation energy is harmlessly dissipated as heat, a process known as qE. We investigated the single-molecule fluorescence intermittency of the main component of the PSII antenna (LHCII) under conditions that mimic efficient use of light or qE, and we demonstrate that weakly fluorescing states are stabilized under qE conditions. Thus, we propose that qE is explained by biological control over the intrinsic dynamic disorder in the complex-the frequencies of switching establish whether the population of complexes is unquenched or quenched. Furthermore, the quenched states were accompanied by two distinct spectral signatures, suggesting more than one mechanism for energy dissipation in LHCII.

THE REACTION CENTER OF PHOTOSYSTEM-II STUDIED WITH POLARIZED FLUORESCENCE SPECTROSCOPY

Abstract Low-temperature steady-state emission properties have been analyzed of Photosystem II reaction center (RC) complexes isolated from spinach CP47-RC complexes after a short Triton X-100 treatment and stabilization in n-dodecyl β, d -maltoside. Excitation spectra of the fluorescence anisotropy were detected at the maximum of the single fluorescence band at 683.5 nm and at the vibrational subband of the same emission at 742 nm. The Q1 transitions of the red-most absorbing pigment(s) showed positive anisotropy with a value of about 0.22. The value is lower than that of the theoretical maximum (0.4) and is explained by a combination of (1) vibrational depolarization effects and (2) by assuming that the red-most absorbing pigments arise from the low-exciton component of P680, that the exciton coupling breaks upon excitation, and that the angle between the monomer Q1 transitions of P680 is 48 ± 10°. The Q1 transitions of pheophytin showed negative anisotropy. This result, combined with the results obtained with linear dichroism spectroscopy, suggests that the spatial organization of the Q1 transitions of pheophytin matches the organization of the bacteriopheophytin residues in the bacterial reaction center. The spatial organization of the y-polarized transitions of pheophytin could be similar in both systems, although these transitions could also be tilted somewhat more towards the membrane plane in PS II. The data furthermore indicate that the accessory chlorophylls in PS II and in bacterial reaction centers have different average orientations, and suggest that at least some of the accessory chlorophylls in PS II have a pheophytin-like orientation.

Multiple Charge‐Separation Pathways in Photosystem II: Modeling of Transient Absorption Kinetics

We explain the transient absorption kinetics (E. Romero, I. H. M. van Stokkum, V. I. Novoderezhkin, J. P. Dekker, R. van Grondelle, Biochemistry 2010, 49, 4300) measured for isolated reaction centers of photosystem II at 77 K upon excitation of the primary donor band (680 nm). The excited-state dynamics is modeled on the basis of the exciton states of 6 cofactors coupled to 4 charge-transfer (CT) states. One CT state (corresponding to charge separation within the special pair) is supposed to be strongly coupled with the excited states, whereas the other radical pairs are supposed to be localized. Relaxation within the strongly coupled manifold and transfer to localized CTs are described by the modified Redfield and generalized Förster theories, respectively. A simultaneous and quantitative fit of the 680, 545, and 460 nm kinetics (corresponding to respectively the Q(y) transitions of the red-most cofactors, Q(x) transition of pheophytin, and pheophytin anion absorption) enables us to define the pathways and time scales of primary electron transfer. A consistent modeling of the data is only possible with a Scheme where charge separation occurs from both the accessory chlorophyll and from the special pair, giving rise to fast and slow components of the pheophytin anion formation, respectively.

Carotenoid Photoprotection in Artificial Photosynthetic Antennas

A series of phthalocyanine-carotenoid dyads in which a phenylamino group links a phthalocyanine to carotenoids having 8-11 backbone double bonds were examined by visible and near-infrared femtosecond pump-probe spectroscopy combined with global fitting analysis. The series of molecules has permitted investigation of the role of carotenoids in the quenching of excited states of cyclic tetrapyrroles. The transient behavior varied dramatically with the length of the carotenoid and the solvent environment. Clear spectroscopic signatures of radical species revealed photoinduced electron transfer as the main quenching mechanism for all dyads dissolved in a polar solvent (THF), and the quenching rate was almost independent of carotenoid length. However, in a nonpolar solvent (toluene), quenching rates displayed a strong dependence on the conjugation length of the carotenoid and the mechanism did not include charge separation. The lack of any rise time components of a carotenoid S(1) signature in all experiments in toluene suggests that an excitonic coupling between the carotenoid S(1) state and phthalocyanine Q state, rather than a conventional energy transfer process, is the major mechanism of quenching. A pronounced inhomogeneity of the system was observed and attributed to the presence of a phenyl-amino linker between phthalocyanine and carotenoids. On the basis of accumulated work on various caroteno-phthalocyanine dyads and triads, we have now identified three mechanisms of tetrapyrrole singlet excited state quenching by carotenoids in artificial systems: (i) Car-Pc electron transfer and recombination; (ii)(1) Pc to Car S(1) energy transfer and fast internal conversion to the Car ground state; (iii) excitonic coupling between (1)Pc and Car S(1) and ensuing internal conversion to the ground state of the carotenoid. The dominant mechanism depends upon the exact molecular architecture and solvent environment. These synthetic systems are providing a deeper understanding of structural and environmental effects on the interactions between carotenoids and tetrapyrroles and thereby better defining their role in controlling natural photosynthetic systems.

Fluorescence Intermittency from the Main Plant Light-Harvesting Complex: Sensitivity to the Local Environment

The time-resolved fluorescence intensity fluctuations from single, immobilized complexes of the main light-harvesting complex (LHCII) of plants were investigated in different pH environments close to room temperature and under different light conditions. The efficiency of light harvesting, which was represented by complexes typically residing for long periods in strongly fluorescing states, was significantly reduced by decreasing the pH or increasing the incident photon flux. The same environmental changes significantly increased the switching frequency between strongly and weakly fluorescing states. The environmental dependence became more evident when the various accessed intensity levels were first resolved, a technique that significantly reduced the obscuring effect of shot noise. The strong environmental sensitivity suggests that the immediate environment of an LHCII complex can modulate the amount of energy dissipation. A simple model illustrates how this may be achieved: the dynamic equilibrium between the strongly and weakly fluorescing states can be shifted by environmentally controlling the conformational diffusion on the potential energy surface of LHCII.

Ultrafast energy transfer in LHC-II trimers from the Chl a b light-harvesting antenna of Photosystem II

Time-resolved absorption difference profiles were obtained for LHC-II trimers, isolated from Photosystem II in spinach with n-dodecyl β-d-maltoside, using one-color and two-color pump-probe techniques. The one-color isotropic signals are predominantly excited state absorption at 640 nm, and a combination of photobleaching and stimulated emission at wavelengths ≥ 665nm. At intermediate wavelengths, dynamic red-shifting due to downhill energy transfer among the chlorophyll (Chl) spectral forms produces a bipolar signal, in which prompt photo-bleaching/stimulated emission is superseded at later times by excited state absorption. Triexponential analyses of these profiles yield the lifetime components 2–6 ps (associated with the spectral shifting), 14–36 ps (possibly due to energy transfer between LHC-II monomers), and several hundred picoseconds. The one-color anisotropy decays are resolvable at 665–675 nm, with lifetimes of 4.3 to 6.3 ps. They are unresolvably fast (i.e., exhibit subpicosecond lifetimes) at 640–650 nm. The two-color isotropic absorption difference signals show clear spectral evolution arising from equilibration among the LHC-II spectral components for excitation wavelengths shorter than 670 nm. However, most of this spectral evolution occurs within less than 2.5 ps. No resolvable anisotropy decay was observed in the two-color experiments. Taken together, the one-color and two-color experiments indicate that both picosecond and subpicosecond energy transfer steps occur in this antenna. The faster processes appear to dominate the spectral equilibration; slower processes occur in isoenergetic energy transfers among the longer-wavelength Chl a spectral forms that absorb between 665 and 675 nm. The values of the long-time anisotropic r(x), measured in the one-color and two-color experiments, are qualitatively consistent with static linear dichroism spectra of these preparations.

Primary processes and structure of the Photosystem II reaction center: II. Low-temperature picosecond fluorescence kinetics of a D1-D2-cyt-b-559 reaction center complex isolated by short Triton exposure

Abstract The fluorescence kinetics of a D 1 -D 2 -cyt- b -559 reaction center complex isolated by short Triton-exposure has been measured with picosecond resolution as a function of temperature below 150 K. The data were analyzed by combined global analysis of data measured with different time resolutions and are presented as decay-associated fluorescence spectra (DAS). Emphasis is given to the resolution and assignment of the fast components below 100 ps. Six lifetime components were generally necessary for a good fit of the data over a long time-range. The shortest lifetime of 13–24 ps (depending on temperature) is attributed to an energy transfer from (accessory) Chlorophyll to P680, presumably via pheophytin. A component in the range of 40–70 ps is attributed to energy transfer from pheophytin to P680. These components can only be properly resolved and assigned from measurements at temperatures below about 40 K. An ultrashort component of 1–6 ps, which had been resolved in our previous measurements (Biochim. Biophys. Acta (1991) 1060, 237–244) was not resolved in the particles studied here. We propose that the absence of the ultrashort component in this study is attributable to the larger chlorophyll content of the present short-term Triton-exposed D 1 -D 2 -cyt- b -559 preparation as compared to the previous long-term Triton-exposed RC particles. In the particles studied here the effective charge separation kinetics in the short time-range is rate-limited by relatively slow energy transfer components (lifetimes from 13 to 24 ps) upon preferential excitation of the accessory chlorophylls. In the long time-range a multicomponent charge recombination kinetics giving rise to four fluorescence lifetimes in the range of 1.56–49.7 ns are resolved. All these components show basically identical DAS with maxima near 682 nm.

Mixed exciton–charge-transfer states in photosystem II: Stark spectroscopy on site–directed mutants

We investigated the electronic structure of the photosystem II reaction center (PSII RC) in relation to the light-induced charge separation process using Stark spectroscopy on a series of site-directed PSII RC mutants from the cyanobacterium Synechocystis sp. PCC 6803. The site-directed mutations modify the protein environment of the cofactors involved in charge separation (P(D1), P(D2), Chl(D1), and Phe(D1)). The results demonstrate that at least two different exciton states are mixed with charge-transfer (CT) states, yielding exciton states with CT character: (P(D2)(δ)(+)P(D1)(δ)(-)Chl(D1)) (673 nm) and (Chl(D1)(δ)(+)Phe(D1)(δ)(-)) (681 nm) (where the subscript indicates the wavelength of the electronic transition). Moreover, the CT state P(D2)(+)P(D1)(-) acquires excited-state character due to its mixing with an exciton state, producing (P(D2)(+)P(D1)(-))(δ) (684 nm). We conclude that the states that initiate charge separation are mixed exciton-CT states, and that the degree of mixing between exciton and CT states determines the efficiency of charge separation. In addition, the results reveal that the pigment-protein interactions fine-tune the energy of the exciton and CT states, and hence the mixing between these states. This mixing ultimately controls the selection and efficiency of a specific charge separation pathway, and highlights the capacity of the protein environment to control the functionality of the PSII RC complex.

Energy-transfer dynamics in three light-harvesting mutants of Rhodobacter sphaeroides: a picosecond spectroscopy study.

Picosecond absorption spectroscopy has been used to investigate energy-transfer dynamics within the LH1 and LH2 light-harvesting complexes of three mutants of Rhodobacter sphaeroides. We demonstrate that both complexes are inhomogeneous; each contains a specialized pigment pool which absorbs maximally at a longer wavelength. Within LH2 (mutant NF57), Bchl850 transfers energy to Bchl870 in 39 +/- 9 ps; within LH1 (mutants M21 and M2192), energy is transferred from Bchl875 to Bchl896 in 22 +/- 4 and 35 +/- 5 ps, respectively. Examination of the decay of induced absorption anisotropy indicates that each of these specialized pools exists in a state which is highly organized with respect to the remainder of the pigments. Such an arrangement may facilitate and direct energy migration toward the reaction center.