H.T. Witt

Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution

Oxygenic photosynthesis is the principal energy converter on earth. It is driven by photosystems I and II, two large protein–cofactor complexes located in the thylakoid membrane and acting in series. In photosystem II, water is oxidized; this event provides the overall process with the necessary electrons and protons, and the atmosphere with oxygen. To date, structural information on the architecture of the complex has been provided by electron microscopy of intact, active photosystem II at 15–30 Å resolution, and by electron crystallography on two-dimensional crystals of D1-D2-CP47 photosystem II fragments without water oxidizing activity at 8 Å resolution. Here we describe the X-ray structure of photosystem II on the basis of crystals fully active in water oxidation. The structure shows how protein subunits and cofactors are spatially organized. The larger subunits are assigned and the locations and orientations of the cofactors are defined. We also provide new information on the position, size and shape of the manganese cluster, which catalyzes water oxidation.

Nanosecond reduction kinetics of photooxidized chlorophyll-aII (P-680) in single flashes as a probe for the electron pathway, H+-release and charge accumulation in the O2-evolving complex☆

(1) The re-reduction kinetics of chlorophyll a+II (P-680+) after the first, second, third etc. flash given to dark-adapted subchloroplasts have been monitored at 824 nm in the nanosecond range. After the first flash and, again, after the fifth flash, the re-reduction of chlorophyll a+II (Chl a+II) in the nanosecond range is nearly monophasic with t12 ≈ 23 ns. After the second and third flash, the re-reduction is significantly slower and biphasic; it can be well-adapted with t12 ≈ 50 ns and ≈260 ns. After the 4th flash, the re-reduction kinetics of Chl a+II are intermediate between the first/fifth and second/third flash. A similar dependence on flash number was obtained with a sample of oxygen-evolving Photosystem II particles from Synechococcus sp. (2) Considering the populations of the S-states of the O2-evolving complex before each flash, the following correlation of S-states to Chl a+II reduction kinetics and electron transfer times, respectively, is obtained: in state S0 as well as in state S1 Chl a+II is reduced with t12 ≈ 23 ns, whereas in state S2 as well as state S3 a biphasic reduction with t12 ≈ 50 ns and ≈260 ns (ratio of the amplitudes ≈1:1) occurs. (3) The observed multiphasic Chl a+II reduction under repetitive excitation is quantitatively explained by a superposition of the individual electron transfer times. (4) We suggest that the retardation of electron transfer to Chl a+II in states S2 and S3 as compared to S0 and S1 is caused by Coulomb attraction by one positive charge located in the O2-evolving complex. A positively charged O2-evolving complex in states S2 and S3 can be explained if the electron release pattern (1,1,1,1) is accompanied by a proton release pattern (1,0,1,2) for the transitions (S0 → S1, S1 → S2, S2 → S3, S3 → S0). (5) A kinetic model based on linear electron transfer from the O2-evolving complex (S) to Chl a+II via two carriers, D1 and D2, makes a quantitative description of the experimental results possible. (6) According to the kinetic model, the retardation of electron transfer to Chl a+II in states S2 and S3 is reflected by an increase in the change of standard free energy, ΔG0, of the reaction Chl a+IID1D2SChl aIID1+D2S from ΔG0 ≈ − 90 meV in states S0 and S1 to ΔG0 ≈ − 20 meV in states S2 and S3. (7) This increase by ≈ 70 meV can be quantitatively explained by the Coulomb potential of the positive charge in the O2-evolving complex, estimated by using the point charge approximation.

Conformational change of the chloroplast ATPase induced by a transmembrane electric field and its correlation to phosphorylation

Abstract The energy-dependent release of bound [14C]nucleotides trom the chloroplast coupling factor CF1, has been used to monitor conformational changes in CF1. The following results were obtained: 1. (1) Similar as in continuous light conformational changes of CF1 are observed on energetization of the thylakoid membrane by short light pulses. Under these conditions the transmembrane electric potential difference induced is about 200 mV and the pH gradient set up across the membrane is about 1.0. 2. (2) Conformational changes are observed also in the dark when external voltage pulses are used for energization. Under these conditions the transmembrane electric potential difference induced is about 200 mV whereas the pH gradient between the inner and outer thylakoid space is zero. 3. (3) Only a fraction of the total number of coupling factors change their conformation. The size of this fraction depends non-linearly on the magnitude of the electric potential difference induced by light pulses or external voltage pulses. 4. (4) In a light or a voltage pulse of 30-ms duration, the amount of ATP generated is 5–8 times larger than the amount of CF1 which have changed their conformation. This factor is independent of the magnitude of the electric potential difference. If the observed conformational changes are coupled with phosphorylation these results may be explained tentatively by the following concept. The proton flux which is used for phosphorylation is focussed only to a fraction of the total number of ATPases. This fraction varies strongly with the electric potential difference (and probably also with the pH gradient). The variation occurs in such a way that the flux via these “active” ATPases and their turnover time is nearly constant (about 5 ms).

Evidence for a trimeric organization of the photosystem I complex from the thermophilic cyanobacterium Synechococcus sp.

A photosystem I (PS I) reaction center complex was isolated and purified from the cyanobacterium Synechococcus sp. The complex has a molecular mass of about 600 kDa and contains 120 Chl a molecules per photoactive Chl a I (P‐700). Electron micrographs show that the PS I complex has the shape of a disk with a diameter of about 19 nm and a thickness of 6 nm. Computer analysis reveals that the complex is composed of three similar units.

Membrane-bound ATP synthesis generated by an external electrical field☆

In the primary act of photosynthesis an electric potential difference, A~, is generated across the energy coupling membrane by a light-induced vectorial electron transfer [1,2]. In a consecutive step protolytic reactions with the charges at the outer and the inner membrane surface lead to the formation of a pH gradient, ApH [3]. Through measurements of the relaxation of A~ simultaneously with the formation of ATP quantitative relationships were obtained between both events in respect to the extent, rate and functional unit [4]. This indicates that phosphorylation is coupled with the discharging of the electrically energized membrane. A coupling of ATP formation with the relaxation of ApH was first demonstrated by Jagendorf and Uribe [5]. Regarding the cooperation of A~ and ApH quantitative relations were elaborated in respect to the kinetics of ATP synthesis [6]. In respect to the energetics there is accumulating evidence that the free energy, AG, stored in A~ ~ 100 mV [7] and ApH ~ 3 [8,9] is with H÷/ATP ~ 2.5 [6,10] in agreement with data of AG [ 11 ] necessary for ATP synthesis. These and other results support the electrochemical hypothesis of Mitchell [12]. Under natural conditions electron transfer, field generation and ApH formation are always coupled with each other. Therefore, with respect to the mechanism of

Size, shape and mass of the oxygen-evolving photosystem II complex from the thermophilic cyanobacterium Synechococcus sp

Two different, highly active O2‐evolving photosystem II complexes were purified from the cyanobacterium Synechococcus sp. in the presence of the non‐ionic detergent β‐dodecyl‐D‐maltoside. Both complexes are homogeneous and have molecular masses of approx. 300 and 500 kDa, respectively. By electron microscopy it was found that both complexes have the shape of an elliptical disk, with a thickness of about 6.5 nm and top view dimensions of 10.5 × 15.5 nm for the 300 kDa particle and 18.5 × 15 nm for the 500 kDa particle. It is concluded that the particles represent monomeric and dimeric forms of photosystem II.

IMPROVED ISOLATION AND CRYSTALLIZATION OF PHOTOSYSTEM I FOR STRUCTURAL ANALYSIS

Abstract Stable trimeric Photosystem I (PS I) was isolated from the cyanobacterium Synechococcus elongatus by use of stereochemically pure β-dodecylmaltoside. Crystals of extremely pure PS I are grown by dialysis against low salt concentration. The improved PS I crystals were the basis for a PS I model derived from X-ray structure analysis at 4 A resolution (Kraus et al., nat. Struct. Biol. 3 (1996) 965–973; Schubert et al., J. Mol. Biol. 272 (1997) 741–769) and for detailed information on the electron transfer chain elaborated by techniques of magnetic resonance (H. Kas, Thesis, TU-Berlin, 1995; Kamlowski et al., Biochim. Biophys. Acta 1319 (1997) 188–198 and 199–213; Bittl et al., Biochemistry 36 (1997) 12001–12004). Crystallization procedures using micro- and macroseeding are described as basis for the forthcoming structure analysis.

Refined purification and further characterization of oxygen-evolving and Tris-treated Photosystem II particles from the thermophilic Cyanobacterium synechococcus sp.

Highly active, monomeric and dimeric Photosystem II complexes were purified from the thermophilic cyanobacterium Synechococcus sp. by two sucrose density gradients, and the size, shape and mass of these complexes have been estimated (Rogner, M., Dekker, J.P., Boekema, E.J. and Witt, H.T. (1987) FEBS Lett. 219, 207–311). (1) Further purification could be obtained by ion-exchange chromatography, by which the 300 kDa monomer could be separated into a highly active, O2-evolving fraction, and a fraction without O2-evolving capacity, which has lost its extrinsic 34 kDa protein. Both showed very high reaction center activities as measured by the photoreduction of the primary quinone acceptor, QA, at 320 nm, being up to one reaction center per 31 Chl a molecules. (2) Tris-treatment yielded homogeneous 300 kDa particles which had lost their extrinsic 34 kDa polypeptide. Electron microscopy of this complex revealed very similar dimensions compared to the oxygen-evolving 300 kDa particle, except that the smallest dimension was decreased from about 6.5 nm to about 5.8 nm. This difference is attributed to the missing extrinsic 33 kDa protein, and the smallest dimension is attributed to the distance across the membrane. (3) Experiments are presented, allowing an estimation for the contribution of detergent to the other dimensions being about 2 × 1.5 nm for dodecyl β-d-maltoside. This leads to dimensions, corrected for detergent size, of 12.3 × 7.5 nm for the monomeric form of PS II and 12 × 15.5 nm for the dimeric form. (4) From some extracts a 35 kDa, chlorophyll-binding complex could be isolated which lacks the characteristic absorbance changes of QA and of Chl aII (P-680) and is therefore supposed to be a light-harvesting complex of cyanobacteria. (5) A model for the in vivo organization of PS II in cyanobacteria is discussed.

Optical characterization of the immediate electron donor to chlorophyll a+II in O2-evolving photosystem II complexes Tyrosine as possible electron carrier between chlorophyll aII and the water-oxidizing manganese complex

The number and chemical nature of the electron carrier(s) between Chl a II and the water‐oxidizing enzyme, S, were analyzed through flash‐induced absorption changes in the UV with nanosecond time resolution. (i) At all wavelengths where the reaction of the donor with Chl a + II has been characterized, this donor is oxidized in the nanosecond time range in exact accordance with the reduction kinetics of Chl a + II. The donor is in turn re‐reduced with t > 10,μs, i.e. in the range where S is oxidized. From this time course it is concluded that there exists only one electron carrier between Chl a + II and S. (ii) The UV‐diference spectrum due to the electron transfer from the immediate donor to Chl a + II in the nanosecond time range in O2‐evolving PS II complexes is characterized by a maximum around 260 nm and smaller minimum around 310 nm. This spectrum is identical with that observed for the reaction of the donor with Chl a + II in the microsecond time range in Tris‐treated PS II. Therefore, the donors in both reactions must be of the same chemical nature. (iii) This result, together with the well‐established similarity of EPR signal IIf of the oxidized donor in Tris‐treated PS II to the EPR signal IIIs, recently assigned to Tyr‐160 of the D2 protein of PS II [(1988) Proc. Natl. Acad. Sci. USA 85, 427–430], provides strong evidence that the immediate donor to Chl a + II in water‐oxidizing PS II is also a tyrosine. (iv) It is shown that the UV‐difference spectra of the oxidation of the immediate donor in O2‐evolving as well as that of Tris‐treated PS II complexes are similar to the in vitro difference spectrum of the oxidation of tyrosine in water. This independent result supports the conclusion that the donor is a tyrosine.

Stoichiometry of Proton Release from the Catalytic Center in Photosynthetic Water Oxidation REEXAMINATION BY A GLASS ELECTRODE STUDY AT pH 5.5–7.2

The catalytic center (CC) of water oxidation in photosystem II passes through four stepwise increased oxidized states (S0–S4) before O2 evolution takes place from 2H2O in the S4 → S0 transition. The pattern of the release of the four protons from the CC cannot be followed directly in the medium, because proton release from unknown amino acid residues also takes place. However, pH-independent net charge oscillations of 0:0:1:1 in S0:S1:S2:S3 have been considered as an intrinsic indicator for the H+ release from the CC. The net charges have been proposed to be created as the charge difference between electron abstraction and H+release from the CC. Then the H+ release from the CC is 1:0:1:2 for the S0 → S1 → S2 → S3 → S0 transition. Strong support for this conclusion is given in this work with the analysis of the pH-dependent pattern of H+ release in the medium measured directly by a glass electrode between pH 5.5 and 7.2. Improved and crystallizable photosystem II core complexes from the cyanobacterium Synechococcus elongatus were used as material. The pattern can be explained by protons released from the CC with a stoichiometry of 1:0:1:2 and protons from an amino acid group (pK ≈ 5.7) that is deprotonated and reprotonated through electrostatic interaction with the oscillating net charges 0:0:1:1 in S0:S1:S2:S3. Possible water derivatives that circulate through the S states have been named.