H. van Amerongen

Chlorophyll a and carotenoid triplet states in light-harvesting complex II of higher plants

Laser-flash-induced transient absorption measurements were performed on trimeric light-harvesting complex II to study carotenoid (Car) and chlorophyll (Chl) triplet states as a function of temperature. In these complexes efficient transfer of triplets from Chl to Car occurs as a protection mechanism against singlet oxygen formation. It appears that at room temperature all triplets are being transferred from Chl to Car; at lower temperatures (77 K and below) the transfer is less efficient and chlorophyll triplets can be observed. In the presence of oxygen at room temperature the Car triplets are partly quenched by oxygen and two different Car triplet spectral species can be distinguished because of a difference in quenching rate. One of these spectral species is replaced by another one upon cooling to 4 Ki demonstrating that at least three carotenoids are in close contact with chlorophylls. The triplet minus singlet absorption (T-S) spectra show maxima at 504-506 nm and 517-523 nm, respectively. In the Chl Qy region absorption changes can be observed that are caused by Car triplets. The T-S spectra in the Chl region show an interesting temperature dependence which indicates that various Cars are in contact with different Chl a molecules. The results are discussed in terms of the crystal structure of light-harvesting complex II.

Singlet-singlet annihilation kinetics in aggregates and trimers of LHCII

Singlet-singlet annihilation experiments have been performed on trimeric and aggregated light-harvesting complex II (LHCII) using picosecond spectroscopy to study spatial equilibration times in LHCII preparations, complementing the large amount of data on spectral equilibration available in literature. The annihilation kinetics for trimers can well be described by a statistical approach, and an annihilation rate of (24 ps)(-1) is obtained. In contrast, the annihilation kinetics for aggregates can well be described by a kinetic approach over many hundreds of picoseconds, and it is shown that there is no clear distinction between inter- and intratrimer transfer of excitation energy. With this approach, an annihilation rate of (16 ps)(-1) is obtained after normalization of the annihilation rate per trimer. It is shown that the spatial equilibration in trimeric LHCII between chlorophyll a molecules occurs on a time scale that is an order of magnitude longer than in Photosystem I-core, after correcting for the different number of chlorophyll a molecules in both systems. The slow transfer in LHCII is possibly an important factor in determining excitation trapping in Photosystem II, because it contributes significantly to the overall trapping time.

Site, rate, and mechanism of photoprotective quenching in cyanobacteria.

In cyanobacteria, activation of the Orange Carotenoid Protein (OCP) by intense blue-green light triggers photoprotective thermal dissipation of excess absorbed energy leading to a decrease (quenching) of fluorescence of the light harvesting phycobilisomes and, concomitantly, of the energy arriving to the reaction centers. Using spectrally resolved picosecond fluorescence, we have studied cells of wild-type Synechocystis sp. PCC 6803 and of mutants without and with extra OCP (ΔOCP and OverOCP) both in the unquenched and quenched state. With the use of target analysis, we managed to spectrally resolve seven different pigment pools in the phycobilisomes and photosystems I and II, and to determine the rates of excitation energy transfer between them. In addition, the fraction of quenched phycobilisomes and the rates of charge separation and quenching were resolved. Under our illumination conditions, ∼72% of the phycobilisomes in OverOCP appeared to be substantially quenched. For wild-type cells, this number was only ∼29%. It is revealed that upon OCP activation, a bilin chromophore in the core of the phycobilisome, here called APC(Q)(660), with fluorescence maximum at 660 nm becomes an effective quencher that prevents more than 80% of the excitations in the phycobilisome to reach Photosystems I and II. The quenching rate of its excited state is extremely fast, that is, at least (∼240 ± 60 fs)(-1). It is concluded that the quenching is most likely caused by charge transfer between APC(Q)(660) and the OCP carotenoid hECN in its activated form.

Probing the many energy-transfer processes in the photosynthetic light-harvesting complex II at 77K using energy-selective sub-picosecond transient absorption spectroscopy.

The dynamics of energy equilibration in the main plant light-harvesting complex, LHCII, at a temperature of 77 K was probed using sub-picosecond excitation pulses at 649, 661, 672 and 682 nm and detection of the resulting difference absorption spectra from 630 to 700 nm. We find three distinct chlorophyll b to chlorophyll a (Chl a) transfer times, of < 0.3, 0.6 and 4–9 ps, respectively. From a comparison of the amplitudes of the bleaching signal, a plausible scheme for the Chl b to Chl a transfer in the LHCII complex is proposed. Two Chl b molecules transfer energy to Chl a in less than 0.3 ps, two Chl b molecules transfer with 0.6 ps and one Chl b has a transfer time of 4–9 ps. In the Chl a absorption region, a 2.4 ps energy-transfer process from a pigment absorbing around 661 nm, and a 0.4 ps process from a pigment absorbing around 672 nm is found. Furthermore, evidence is found for slow, 10–20 ps energy-transfer processes between some of the Chl a molecules. The data are compared to model calculations using the 3.4 A LHCII monomer structure (containing 5 Chl b and 7 Chl a molecules) and Forster energy transfer. We conclude that the observed energy-transfer rates are consistent with both the preliminary assignment of the Chl identities (a or b) of Kuhlbrandt et al. and a recent proposal for the arrangement of some of the transition dipole moments (Gulen et al.). Singlet-singlet and singlet-triplet annihilation processes are observed in two different experiments, and both these processes occur with time constants of 2–3 and 12–20 ps, suggesting that both annihilation pathways are at least partly limited by slow energy transfer. The wide range of observed time constants in the equilibration, from < 0.3 to ∼ 20 ps, most likely reflects the irregular arrangement of the pigments in the complex, which shows much less symmetry than the recently obtained structure of the peripheral antenna complex of purple bacteria, LH-II (McDermott et al.).

Linear Dichroism of Chlorosomes from Chloroflexus Aurantiacus in Compressed Gels and Electric Fields

The linear dichroism of chlorosomes from Chloroflexus aurantiacus was measured between 250 and 800 nm. To orient the chlorosomes we used a new way of compressing polyacrylamide gels, where the dimension of the gel along the measuring light-beam is kept constant. The press required for such a way of compressing is relatively easy to construct. A theoretical description is given to interpret the measured linear dichroism in terms of the orientation of the transition moments. The results obtained with the polyacrylamide gels are compared with the linear dichroism measurements for chlorosomes oriented in electric fields. Both the spectral features as well as the absolute size of the linear dichroism signals are in reasonable agreement. We find that the transition moment corresponding to the 741 nm bacteriochlorophyll c (Bchl c) absorption band makes an angle of 20 degrees with the long axis of the chlorosome. For the 461 nm Bchl c band an angle of 30 degrees is found. Both angles are significantly lower than the values reported so far in literature and they imply that Bchl c is highly organized in the chlorosomes.

The organization of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus and the structural role of carotenoids and protein. An absorption, linear dichroism, circular dichroism and Stark spectroscopy study.

The organization of bacteriochlorophyll c (BChl c) molecules was studied in normal and carotenoid-deficient chlorosomes isolated from the green phototrophic bacterium Chloroflexus aurantiacus. Carotenoid-deficient chlorosomes were obtained from cells grown in the presence of 60 µg of 2-hydroxybiphenyl per ml. At this concentration, BChl c synthesis was not affected while the formation of the 5.7 kDa chlorosome polypeptide was inhibited by about 50% (M. Foidl et al., submitted). Absorption, linear dichroism and circular dichroism spectroscopy showed that the organization of BChl c molecules with respect to each other as well as to the long axis of the chlorosomes was similar for both types of chlorosomes. Therefore, it is concluded that the organization of BChl c molecules is largely independent on the presence of the bulk of carotenoids as well as of at least half of the normal amount of the 5.7 kDa polypeptide. The Stark spectra of the chlorosomes, as characterized by a large difference polarizability for the ground- and excited states of the interacting BChl c molecules, were much more intense than those of individual pigments. It is proposed that this is caused by the strong overlap of BChl c molecules in the chlorosomes. In contrast to individual chlorophylls, BChl c in chlorosomes did not give rise to a significant difference permanent dipole moment for the ground- and excited states. This observation favors models for the BChl c organization which invoke the anti-parallel stacking of linear BChl c aggregates above those models in which linear BChl c aggregates are stacked in a parallel fashion. The difference between the Stark spectrum of carotenoid-deficient and WT chlorosomes indicates that the carotenoids are in the vicinity of the BChls.

Polarized fluorescence and absorption of macroscopically aligned Light Harvesting Complex II

Polarized absorption and fluorescence measurements have been performed at 77 K on isotropic and anisotropic preparations of trimeric Light Harvesting Complex II (LHC-II) from spinach. The results enable a decomposition of the absorption spectrum into components parallel and perpendicular to the trimeric plane. For the first time, it is shown quantitatively that the strong absorption band around 676 nm is polarized essentially parallel to the plane of the trimer, i.e., the average angle between the corresponding transition dipole moments and this plane is at most 12 degrees. The different absorption bands for LHC-II should not be considered as corresponding to individual pigments but to collective excitations of different pigments. Nevertheless, the average angle between the Qy transition dipole moments of all chlorophyll a pigments in LHC-II and the trimeric plane could be determined and was found to be 17.5 degrees +/- 2.5 degrees. For the chlorophyll b pigments, this angle is significantly larger (close to 35 degrees). At 77 K, most of the fluorescence stems from a weak band above 676 nm and the corresponding transition dipole moments are oriented further out of plane than the dipole moments corresponding to the 676-nm band. The results are shown to be of crucial significance for understanding the relation between the LHC-II structure and its spectroscopy.

Steady-state and time-resolved polarized light spectroscopy of the green plant light-harvesting complex II

Abstract The major chlorophyll a/b light-harvesting complex from spinach thylakoid membranes was analyzed by steady-state polarized light spectroscopy at 4 K and by one-color and two-color pump-probe spectroscopy at room temperature. Steady-state absorption, linear dichroism and circular dichroism spectra indicate that the Chl Q y (0−0) absorption region is characterized by at least six transitions with significant differences in absorption, orientation and rotational strength. Steady-state low-temperature fluorescence spectra suggest that the fluorescence arises for a large part from several energetically similar species that form a circularly degenerate oscillator in the plane constituted by the two long axes of the particle. The possible presence of special red-absorbing pigments at low temperature is discussed. The time-resolved data suggest that the kinetics of chlorophyllb → a excitation energy transfer, as well as those of downhill excitation transfer among chlorophylla spectral forms, are heterogeneous with both sub-picosecond and picosecond lifetime components.

High-precision 2-D SM fiber connectors fabricated through deep proton writing

High-precision two-dimensional (2-D) fiber alignment modules would offer great benefits for high-density photonic interconnects at the multichip-module level, where parallel light signals have to be transferred between integrated dense 2-D emitter and detector arrays, or for massive parallel sensing applications. In telecom, the availability of highly accurate low-cost field installable 2-D fiber couplers would boost the further integration of fiber optics in future fiber-to-the-home networks. We present deep proton writing as a prototyping technology for the mastering of small-form-factor 2-D fiber connector components. The alignment components, which we present here, consist of 4 times 8 arrays of circular conically shaped holes for single-mode fibers and feature average insertion losses of 0.062 dB and a maximum loss of 0.15 dB, when used in a fiber butt-coupling configuration

Spectroscopic characterisation of the reaction centre of photosystem II from higher plants

Two different photosystem II particles isolated from pea, a core complex and the D1/D2/Cyt b‐559 reaction centre (RC) complex, have been characterised by absorption, linear dichroism (LD) and circular dichroism (CD) spectroscopy. Only one carotenoid contributes to the LD of the reaction centre, in agreement with the biochemical analysis. This carotenoid is oriented parallel to the long axis of the reaction centre. The chlorophyll QY contribution to the LD is oriented perpendicular to the long axis of the reaction centre. The LD of the reaction centre carotenoid is reversed in the core complex. In addition, the contribution of a second carotenoid species can be observed. In the core, the two pools of carotenoid have a rather different orientation with respect to the membrane plane: one parallel, the other perpendicular. In addition, in the core the sign of the LD in the chlorophyll QY region is reversed and red‐shifted as compared to the reaction centre. These observations suggest that the reaction centre is oriented with its long axis perpendicular to the long axis of the PS II core and to the membrane plane. The circular dichroism CD of the reaction centre has intense peaks of opposite sign at 444 nm (negative) and 435 nm (positive), which we attribute to exciton coupling between Chl a molecules in the reaction centre. The RC has no CD in the range 460–650 nm; thus there is no exciton coupling between the carotenoid and the other pigments. The lack of CD in this region is consistent with the biochemical analysis of only one carotenoid per reaction centre. The larger core complex exhibits much weaker CD (per Chl). The CD in the QY absorption band indicates exciton coupling between chlorophyll molecules. The sign of the pair of peaks in this region is reversed in the core with respect to the reaction centre.