Determination of the pigment stoichiometry of the photochemical reaction center of photosystem II

AbstractThe stoichiometry of chlorophyll a, pheophytin α and β-carotene in the photochemical reaction center of Photosystem II was analyzed by reversed phase high-performance liquid chromatography (HPLC) with methanol as the mobile phase, and by the shape of spectra of extracts in 80% acetone. For the HPLC method the molar extinction coefficient of pheophytin a in methanol was redetermined, while for the spectroscopic method spectra of extracts in 80% acetone were simulated by fitting with spectra of isolated chlorophyll a, pheophytin a and β-carotene in 80% acetone. Both methods give internally consistent results, and suggest that the reaction center of Photosystem II isolated by a short Triton X-100 treatment binds 6 chlorophyll a per 2 pheophytin a molecules. We also present evidence that prolonged exposure of the Photosystem II reaction center complex to Triton X-100 does not result in the loss of chlorophyll from the complex. Based on a comparison with spectra reported in publications from other groups, we conclude that the chlorophyll to pheophytin ratio has previously been underestimated to sometimes very significant extents, and that, as yet, no Photosystem II reaction center particles have been purified that bind less than 5–6 chlorophyll a per 2 pheophytin a

Purification and spectroscopic characterization of photosystem II reaction center complexes isolated with or without Triton X-100.

The pigment composition of the isolated photosystem II reaction center complex in its most stable and pure form currently is a matter of considerable debate. In this contribution, we present a new method based on a combination of gel filtration chromatography and diode array detection to analyze the composition of photosystem II reaction center preparations. We show that the method is very sensitive for the detection of contaminants such as the core antenna protein CP47, pigment-free and denatured reaction center proteins, and unbound chlorophyll and pheophytin molecules. We also present a method by which the photosystem II reaction center complex is highly purified without using Triton X-100, and we show that in this preparation the contamination with CP47 is less than 0.1%. The results strongly indicate that the photosystem II reaction center complex in its most stable and pure form binds six chlorophyll a, two pheophytin a, and two β-carotene molecules and that the main effect of Triton X-100 is the extraction of β-carotene from the complex. Analysis of 4 K absorption and emission spectra indicates that the spectroscopic properties of this preparation are similar to those obtained by a short Triton X-100 treatment. In contrast, preparations obtained by long Triton X-100 treatment show decreased absorption of the shoulder at 684 nm in the 4 K absorption spectrum and an increased number of pigments that trap excitation energy at very low temperatures. We conclude that the 684 nm shoulder in the 4 K absorption spectrum should at least in part be attributed to the primary electron donor of photosystem II

Characterization by electron microscopy of dimeric Photosystem II core complexes from spinach with and without CP43

Dimeric associations of the D1-D2-CP47 and D1-D2-CP47-CP43 complexes of Photosystem II from spinach were isolated and purified with sucrose density gradient centrifugation and gel filtration chromatography and analyzed by electron microscopy and image analysis. Images of both preparations show characteristic details in protein density. The location of the CP43 subunit and the way the dimers are associated could be determined from a comparison between diamond-like monomeric projections of the D1-D2-CP47-CP43 complex (maximal dimensions along the diagonals 10–12 and 7–8 nm) and triangle-like monomeric projections of the D1-D2-CP47 complex (dimensions 8–9 and 7–8 nm). Both isolated complexes have different dimeric configurations than observed before in several other dimeric complexes, and based on biochemical considerations we conclude that both newly observed configurations are artificial. The observation of the artificial aggregates, however, allows conclusions on the organization of Photosystem II in two-dimensional crystals and on the size of the monomeric unit. We propose a model for the location of D1, D2, CP43 and CP47 in the Photosystem II core complex in which CP43 and CP47 are positioned at the tips of the monomeric unit, closely connected to D2 and D1, respectively.

Two different charge-separation pathways in photosystem II

Charge separation is an essential step in the conversion of solar energy into chemical energy in photosynthesis. To investigate this process, we performed transient absorption experiments at 77 K with various excitation conditions on the isolated Photosystem II reaction center preparations from spinach. The results have been analyzed by global and target analysis and demonstrate that at least two different excited states, (Ch

Spectral hole burning: examples from photosynthesis

The optical spectra of photosynthetic pigment–protein complexes usually show broad absorption bands, often consisting of a number of overlapping, ‘hidden’ bands belonging to different species. Spectral hole burning is an ideal technique to unravel the optical and dynamic properties of such hidden species. Here, the principles of spectral hole burning (HB) and the experimental set-up used in its continuous wave (CW) and time-resolved versions are described. Examples from photosynthesis studied with hole burning, obtained in our laboratory, are then presented. These examples have been classified into three groups according to the parameters that were measured: (1) hole widths as a function of temperature, (2) hole widths as a function of delay time and (3) hole depths as a function of wavelength. Two examples from light-harvesting (LH) 2 complexes of purple bacteria are given within the first group: (a) the determination of energy-transfer times from the chromophores in the B800 ring to the B850 ring, and (b) optical dephasing in the B850 absorption band. One example from photosystem II (PSII) sub-core complexes of higher plants is given within the second group: it shows that the size of the complex determines the amount of spectral diffusion measured. Within the third group, two examples from (green) plants and purple bacteria have been chosen for: (a) the identification of ‘traps’ for energy transfer in PSII sub-core complexes of green plants, and (b) the uncovering of the lowest k = 0 exciton-state distribution within the B850 band of LH2 complexes of purple bacteria. The results prove the potential of spectral hole burning measurements for getting quantitative insight into dynamic processes in photosynthetic systems at low temperature, in particular, when individual bands are hidden within broad absorption bands. Because of its high-resolution wavelength selectivity, HB is a technique that is complementary to ultrafast pump–probe methods. In this review, we have provided an extensive bibliography for the benefit of scientists who plan to make use of this valuable technique in their future research

A routine method to determine the chlorophyll a, pheophytin a and B-carotene contents of isolated photosystem II reaction center complexes.

The most simple way in which the stoichiometry of chlorophyll a, pheophytin a and ̄-carotene in isolated Photosystem II reaction center complexes can be determined is by analysis of the spectrum of the extracted pigments in 80% acetone. We present two different calculation methods using the extinction coefficients of the purified pigments in 80% acetone at different wavelengths. One of these methods also accounts for the possible presence of chlorophyll b. The results are compared with results obtained with HPLC pigment analysis, and indicate that these methods are suitable for routine determination of the pigment stoichiometry of isolated Photosystem II reaction center complexes

Specific association of photosystem II and light-harvesting complex II in partially solubilized photosystem II membranes.

AbstractIn this study, we report the structural characterization of photosystem II complexes obtained from partially solubilized photosystem II membranes. Direct observation by electron microscopy, within a few minutes after a mild disruption of the membranes with the detergent n-dodecyl-α,d-maltoside, revealed the presence of several large supramolecular complexes. Images of these complexes were subjected to multivariate statistical analysis and classification procedures, resolving a new complex consisting of the previously characterized dimeric supercomplex of photosystem II and light-harvesting complex II [Boekema et al., Proc. Natl. Acad. Sci. USA 92 (1995) 175–179] and two additional, symmetrically organized protein masses each containing a second type of trimeric light-harvesting II complex. We conclude that large and labile integral membrane proteins, such as photosystem II, can be quickly structurally characterized without extensive purification

Spectroscopic characterization of a 5 Chl a photosystem II reaction center complex.

AbstractIt is now well established that the isolated photosystem II (PS II) reaction center complex in its most stable form binds 6 chlorophyll a, 2 β-carotene and 2 pheophytin a molecules. By using immobilised metal affinity chromatography, however, it is possible to isolate PS II reaction center particles binding 5 Chl a molecules [Vacha et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2929–2933]. In this report we present a number of steady-state spectroscopic characteristics at very low temperature(s) of the 5 Chl preparation (RC-5) and compare those with data obtained for 6 Chl preparations (RC-6). The results confirm the loss of a chlorophyll molecule absorbing at 670 nm in RC-5, and in addition reveal that the shoulder near 684 nm is more pronounced in this preparation than in any other PS II RC preparation. The RC-5 preparation is therefore ideally suited to obtain more information on the nature of the low-energy absorption. Based on the fluorescence and triplet-minus-singlet absorbance-difference data presented in this paper, we propose that all absorption around 680 and 684 nm arises from the weakly excitonically coupled `core' of the RC-5 complex, and that the remaining peripheral Chl molecule absorbs at 670 nm. Furthermore, from the temperature dependence of the spectroscopic data we conclude that the 684 nm absorption in isolated PS II reaction center complexes contains about equal contributions from the primary electron donor and from the red-absorbing `trap' states