Purification and Spectroscopic Characterization of Photosystem II Reaction Center Complexes Isolated with or without Triton X-100

Eijckelhoff, Camiel; Roon, Henny van; Groot, Marie Louise; Grondelle, Rienk van; Dekker, Jan

doi:10.1021/bi960991m

Cited by 37 publications

(29 citation statements)

References 35 publications

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“…Therefore, the onset of acceleration of the average energy transfer time as a function of temperature may be expected to occur at lower temperature. To explain the acceleration from 90 ps to 35 ps, assuming an Arrhenius type of expression, at 77 K a change in activation energy of only 49 cm Ϫ1 or 2.2 nm is needed, which is consistent with the observed narrowing (38). Although the variation of the samples is discomforting, it lends support for the notion that the observed temperature behavior of the kinetics originates from the inhomogeneity of two degenerate electronic transitions.…”

Section: Discussionsupporting

confidence: 62%

“…They are characterized by a deeper valley between the main bands in the 4 K absorption spectrum, and by a more pronounced shoulder at 684 nm than observed for the preparations used in the earlier experiments (31). In Eijckelhoff et al (38) this subtle change in the Q y region is explained by a narrowing of the underlying absorption bands in the red part of the spectrum, from Ϸ6 nm to Ϸ4 nm. A narrowing of the inhomogeneous absorption bands of both P680 and the trap pigments will lead to a decrease in the average activation energy between the trap pigments and P680.…”

Section: Discussionmentioning

confidence: 98%

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Charge separation in the reaction center of photosystem II studied as a function of temperature

Groot

Mourik

Eijckelhoff

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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In photosystem II of green plants the key photosynthetic reaction consists of the transfer of an electron from the primary donor called P680 to a nearby pheophytin molecule. We analyzed the temperature dependence of this reaction by subpicosecond transient absorption spectroscopy over the temperature range 20-240 K using isolated photosystem II reaction centers from spinach. After excitation in the red edge of the Q y absorption band, the decay of the excited state can conveniently be described by two kinetic components that both accelerate with temperature. This temperature behavior differs remarkably from that observed in purple bacterial reaction centers. We attribute the first component, which accelerates from 2.6 ps at 20 K to 0.4 ps at 240 K, to charge separation after direct excitation of P680, and explain its temperature dependence by an intermediate that lies in energy above the singlet-excited P680 and that possibly has charge-transfer character. The second component accelerates from 120 ps at 20 K to 18 ps at 240 K and is attributed to charge separation after direct excitation of the ''trap'' state near-degenerate with P680 and subsequent slow energy transfer from this trap state to P680. We suggest that the slow energy transfer from the trap state to P680 plays an important role in the kinetics of radical pair formation at room temperature.The photochemical reaction center (RC) of photosystem II (PSII) (the D1-D2 cyt.b559 complex) is the smallest unit in PSII that shows photochemical activity. The RC contains six chlorophyll (Chl) a and two pheophytin (Pheo) a molecules (1-4) that all have their lowest electronic transition around 675 nm, as well as two ␤-carotenes. The D1 and D2 polypeptides are homologous to the L and M subunits of bacterial RCs, suggesting an arrangement of the core pigments in the RC of PSII similar to that in the bacterial RC (5). The two additional Chl molecules are probably located near the periphery of the D1-D2 complex.The characterization of energy transfer and charge separation in the PSII RC is less well established than is the case for the bacterial RC (for a review, see ref. 6). This may be due to the fact that the first PSII RC was isolated only in 1987 (7) and that there is no structure available. However, the excited state kinetics of the PSII RC are also inherently more complicated than those of bacterial RCs. The primary electron donor in PSII, called P680, is isoenergetic with some of the other pigments in the RC (8-13) and as a consequence forms only a very shallow trap for excitations.After excitation of P680 an electron is transferred to the photoactive Pheo molecule (6). In isolated PSII RC preparations, further electron transport cannot occur, because the quinone acceptors are lost during the isolation procedure. A straightforward interpretation of the results from pump-probe measurements in terms of pigment to pigment energy transfer and charge separation rates has proven difficult, mainly due to the congested absorption spectrum of the PSII RC. At r...

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Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 98%

Charge separation in the reaction center of photosystem II studied as a function of temperature

Groot

Mourik

Eijckelhoff

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…We subjected PS II membranes to a short treatment of detergent in quantities less than required to completely solubilize the membranes. With the gel ¢ltration techniques developed and applied for PS II RC complexes in [10], we found with L-DM as detergent several fractions with clearly di¡erent A RUH / A RQS ratios (Fig. 1A), indicating the presence several types of complexes with very di¡erent Chl b/Chl a ratios.…”

Section: Resultsmentioning

confidence: 99%

“…The suspension was stirred during 5 s, centrifuged during 3 min at 9000 rpm in an Eppendorf 5414 table centrifuge and pushed through a 0.45 Wm ¢lter to remove unsolubilized material, after which it was immediately used for further analysis. The composition of the solubilized material was checked by gel ¢ltration chromatography using a Superdex 200 HR 10/30 column (Pharmacia) and diode array detection as described in [10]. In the mobile phase, however, 0.03% L-DM was replaced by 0.03% K-DM when K-DM was used as the solubilizing agent.…”

Section: Methodsmentioning

confidence: 99%

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

1998

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In 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-K K,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.z 1998 Federation of European Biochemical Societies.

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“…32. Anaerobic conditions were achieved by bubbling the D 2 O buffer solution (pH 6.5) with argon gas for 2 h. The samples were concentrated to an OD of 0.2 per 20 m at 675 nm.…”

Section: Methodsmentioning

confidence: 99%

Initial electron donor and acceptor in isolated Photosystem II reaction centers identified with femtosecond mid-IR spectroscopy

Groot

Pawlowicz

Wilderen

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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258

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Despite the apparent similarity between the plant Photosystem II reaction center (RC) and its purple bacterial counterpart, we show in this work that the mechanism of charge separation is very different for the two photosynthetic RCs. By using femtosecond visible-pump-mid-infrared probe spectroscopy in the region of the chlorophyll ester and keto modes, between 1,775 and 1,585 cm ؊1 , with 150-fs time resolution, we show that the reduction of pheophytin occurs on a 0.6-to 0.8-ps time scale, whereas P ؉ , the precursor state for water oxidation, is formed after Ϸ6 ps. We conclude therefore that in the Photosystem II RC the primary charge separation occurs between the ''accessory chlorophyll'' ChlD1 and the pheophytin on the so-called active branch.electron transfer ͉ photosynthesis ͉ pump-probe T he primary steps of energy and electron transfer in green plants' photosynthesis occur in two large protein complexes called Photosystem I and Photosystem II (PSII). PSII is an aggregate of many individual pigment-protein complexes. The core of PSII consists of the chlorophyll (Chl)-binding antennaproteins CP43 and CP47, which feed excitation energy into the D 1 D 2 cytb559 reaction center (RC). Crystal structures of PSII cores from cyanobacteria have been resolved with increasingly high resolution (1-3); currently, the resolution is 3.2 Å (4). The structure of the PSII RC shows four Chls and two pheophytins (H) arranged in two branches very similar to the bacterial RC. In the heart of the PSII RC, there is a dimer of Chls, and in each branch there is one monomeric Chl and one H. Furthermore, there are two distant Chls bound to the periphery of the PSII RC. Although the structure suggests there may be a ''special pair'' of strongly electronically coupled pigments in the PSII RC, the visible absorption spectrum does not show a distinct band. This finding is in contrast to the bacterial RC, where the lowest energy absorption band fully originates from one of the exciton transitions of a special pair of bacteriochlorophylls.Since the first purification of the PSII RC in 1987 (5), it has been speculated that its way of operation would be similar to that of the bacterial RC, with a special pair that upon excitation drives a charge separation in Ϸ3 ps. This idea was based on the strong homology between the bacterial RC and the PSII RC, the strong similarity in the pigment composition, even details in the way the pigments interacted with the protein, and the near-identity of the electron transfer events at the acceptor side. Conversely, it was clear that major differences between the two RCs had to exist at the electron donor side where in the PSII RC charge separation eventually leads to the oxidation of water and the production of molecular oxygen, requiring a very large oxidation potential of the primary electron donor (Ͼ1.2 V vs. 0.45 V in the bacterial RC).In the mid-1990s, it was recognized that energy transfer and charge separation in the PSII RC most likely proceeded in a manner that is very different from that in the b...

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Purification and Spectroscopic Characterization of Photosystem II Reaction Center Complexes Isolated with or without Triton X-100

Cited by 37 publications

References 35 publications

Charge separation in the reaction center of photosystem II studied as a function of temperature

Charge separation in the reaction center of photosystem II studied as a function of temperature

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

Initial electron donor and acceptor in isolated Photosystem II reaction centers identified with femtosecond mid-IR spectroscopy

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