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

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

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

Role of Arg180 of the D2 protein in photosystem II structure and function.

On the basis of sequence comparison with the M subunit of the reaction center of purple bacteria, no residues in photosystem II can be clearly identified that may be predicted to correspond to the His residue that binds one of the accessory bacteriochlorophylls in the purple bacterial reaction center. However, the Arg180 residue of the D2 protein is close to where this residue is predicted to be and could conceivably serve as a chlorophyll ligand. To analyze the function of Arg180, it was changed to nine different amino acids in the cyanobacterium Synechocystis sp. PCC 6803. Except for the Arg180→Gln (R180Q) mutant, the resulting strains were no longer photoautotrophic. The properties of photosystem II upon mutation of Arg180 were probed in strains from which photosystem I had been deleted genetically. Mutations at the Arg180 residue affected oxygen evolution capacity and the amount of photosystem II that was present in thylakoids. Surprisingly, in the Arg180 mutants, EPR signals that may originate from the oxidized redox-active Tyr160 of the D2 protein (Y(D)(ox)) were small and generally did not resemble the usual signal II(s), signifying an effect of the Arg180 mutations on the environment surrounding Tyr160. In addition, in most mutants, the charge recombination kinetics between the primary electron-accepting quinone in photosystem II (Q(A

Two-step sequential light-induced electron transfer in a simple trichromophoric donor-donor-acceptor system

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Synthesis and exploratory photophysical investigation of donor-brige-acceptor systems derived from N-substituted 4-piperidones

We report a two-step synthesis for N-aryl- and N-alkyl-substituted 4-piperidones, in which the N substituent can easily be varied. A number of intramolecular donor-acceptor systems was synthesized from these piperidones by conversion of the carbonyl functionality. The influence of the N-aryl donor on the electronic absorption and fluorescence spectra was investigated systematically. It was concluded that some systems can be used as efficient fluorescent probes with a high sensitivity for solvent polarity