Florentine Calkoen

Characterisation of LHC II in the aggregated state by linear and circular dichroism spectroscopy

Abstract Absorption, linear dichroism and circular dichroism spectroscopy at 293 and 77 K have been used in order to further explore the process of energy quenching in LHC IIb, the main light-harvesting complex of photosystem II. Upon aggregation there was an enhancement of linear dichroism bands in the Q y absorption region of chlorophyll b . The absorption spectrum at 77 K of aggregates revealed new bands around 656 nm and 680 nm, characterised by positive linear dichroism and negative circular dichroism signals. In the circular dichroism spectrum of aggregates a characteristic change was seen in the carotenoid and chlorophyll b regions, an increase of the chlorophyll a transition at 438 nm (−) and decrease of the red most negative band at around 677 nm. The amplitude of this band was in a tight correlation with a fluorescence quenching occurring upon LHC II aggregation. A new transition appeared at 505 nm with positive linear dichroism signal. It is suggested that protein aggregation causes a change in conformation and association of some chlorophyll a , chlorophyll b and xanthophyll molecules. These features of the linear dichroism spectrum of the aggregates were also detected for thylakoids in which they were particularly enhanced at low pH, suggesting that at least part of the light harvesting complex in the thylakoid membrane is in an aggregated form and the extent of aggregation in vivo can be controlled by the thylakoid pH gradient.

Structural characterization of the B800-850 and B875 light-harvesting antenna complexes from Rhodobacter sphaeroides by electron microscopy

Abstract The structure and aggregation behavior of B800–850 (LHII) and B875 (LHI) antenna complexes of Rhodobacter sphaeroides were studied by electron microscopy. Single molecular projections (top views and side views) of isolated particles were analyzed. The B800–850 complexes, isolated as 150 kDa particles, are cylindrical with a diameter of 8.5 nm and with a height of 6.5 nm. If corrected for attached detergent, the actual diameter in the plane of the membrane would be about 5.1 nm. Stain accumulation in the center of the structure indicates a small indentation, therefore a ring-shaped structure is expected. The size, shape and estimated mass give evidence for a B800–850 complex structure consisting of 4–6 αβ heterodimers, if in an αnβn ring-shaped configuration. The B875 complexes, isolated as 360 kDa particles, are dimeric units, but the two monomers are only loosely connected. A single B875 unit has a corrected diameter of about 5.2 nm and a height of 6.2 nm, similar to the B800–850 unit. Therefore, the B875 complex most likely has the same αnβn configuration. Image analysis of B800–850 projections points to a 3-or 6-fold symmetry in the top views, rather than a 4- or 5-fold symmetry. Therefore, our results are most compatible with an α6β6 configuration.

Low-temperature spectroscopy of monomeric and trimeric forms of reconstituted light-harvesting chlorophyll a/b complex.

Low-temperature (polarized) light spectroscopy was used to study reconstituted light-harvesting complex (LHCII) of Photosystem II, both in the monomeric and trimeric form. Monomeric LHCII was reconstituted from the apoprotein overexpressed in Escherichia coli and pigments were extracted from chloroplast membranes and subsequently separated from unbound pigments on an anion-exchange column. These monomers trimerize in the presence of a lipid fraction isolated from thylakoids or of pure phosphatidylglycerol. The spectroscopic properties are compared to those of monomeric and trimeric forms of native LHCII and many similarities exist. However, these reconstituted complexes seem to contain slightly fewer chlorophylls, whereas one pigment that is a chlorophyll a in native LHCII is replaced by a chlorophyll b in reconstituted LHCII.

Reversible Light-Inditced Fluorescence Quenching — An Inherent Property of LHCII

Illumination of light-harvesting chlorophyll a/b pigment-protein complex (LHCII) induces fluorescence quenching which is reversible in the dark. The phenomenon was first observed by Jennings et al. [1] and was proposed to play a role in the non-photochemical quenching of chlorophyll fluorescence in photosynthetic organisms. Light-induced quenching of fluorescence which is not related to the photochemistry appears to be of paramount importance in the adaptation mechanisms of plants to different light intensities and protection of plants from photodamage under excess solar radiation conditions [2].

Spectroscopic Characterization of Reconstituted LHCII Which Contains Mainly CHL B and Xanthophylls.

In [1] the structure of the light-harvesting chlorophyll alb-protein complex, LHCII, was reported at 3.4 A resolution. The trimeric complex revealed twelve chlorophylls (Chls) per monomeric subunit, seven of which were in Van der Waals contact with two central xanthophyll molecules that were supposed to be luteins. The identities of the Chis (Chi a or Chl b) were not clear from the structure but the seven interior Chls next to the xanthophylls were assigned as Chl a. The main argument for this assignment was the following: excitations on Chi can lead to triplet formation on a ns time scale. The xanthophylls provide LHCII with the possibility of actively quenching Chl triplet states if the Chls and xanthophylls are in Van der Waals contact. If they were left unchecked, these triplet states would combine with oxygen molecules to create highly reactive and toxic singlet species. Since excitations on Chi b are being transferred to Chi a on a (sub)picosecond time scale, mainly Chi a triplets need to be quenched. This assignment was of course tentative. However, there was also some experimental support for it. Optically detected magnetic resonance (ODMR) experiments showed that triplets located on the xanthophylls did significantly influence the absorption spectrum of Chl a whereas virtually no effect on the Chl b absorption could be detected [2]. Later this effect was confirmed with the use of laser flash-induced triplet-minus-singlet spectroscopy [3, 4]. Although only contacts between xanthophyll and Chl a could be demonstrated in this way, it could not be ruled out that there might also be some contacts between xanthophyll and Chl b leaving the absorption of Chl b unaffected in the presence of xanthophyll triplets.