Photosystem II single crystals studied by EPR spectroscopy at 94 GHz: the tyrosine radical Y(D)(*).

Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y(D)(*) in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y(D)(*) and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y(D)(*) in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.

Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution.

Life on Earth depends on photosynthesis, the conversion of light energy from the Sun to chemical energy. In plants, green algae and cyanobacteria, this process is driven by the cooperation of two large protein-cofactor complexes, photosystems I and II, which are located in the thylakoid photosynthetic membranes. The crystal structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus described here provides a picture at atomic detail of 12 protein subunits and 127 cofactors comprising 96 chlorophylls, 2 phylloquinones, 3 Fe4S4 clusters, 22 carotenoids, 4 lipids, a putative Ca2+ ion and 201 water molecules. The structural information on the proteins and cofactors and their interactions provides a basis for understanding how the high efficiency of photosystem I in light capturing and electron transfer is achieved.

Crystal structure of photosystem II from Synechococcus elongatus at 3.8 A 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 A resolution, and by electron crystallography on two-dimensional crystals of D1-D2-CP47 photosystem II fragments without water oxidizing activity at 8 A 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.

First photosystem II crystals capable of water oxidation.

Oxygen evolution and proton release of crystallised photosystem II core complexes isolated from Synechococcus elongatus were measured. The yields show that the crystals themselves are capable of highly active water oxidation. This opens the possibility for the structural analysis of the outstanding water-oxidising apparatus.

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 (S(0)-S(4)) before O(2) evolution takes place from 2H(2)O in the S(4) --> S(0) 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 S(0):S(1):S(2):S(3) 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 S(0) --> S(1) --> S(2) --> S(3) --> S(0) 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 approximately 5.7) that is deprotonated and reprotonated through electrostatic interaction with the oscillating net charges 0:0:1:1 in S(0):S(1):S(2):S(3). Possible water derivatives that circulate through the S states have been named.

Photosystem I, an improved model of the stromal subunits PsaC, PsaD, and PsaE.

An improved electron density map of photosystem I (PSI) calculated at 4-A resolution yields a more detailed structural model of the stromal subunits PsaC, PsaD, and PsaE than previously reported. The NMR structure of the subunit PsaE of PSI from Synechococcus sp. PCC7002 (Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062) has been used as a model to interpret the region of the electron density map corresponding to this subunit. The spatial orientation with respect to other subunits is described as well as the possible interactions between the stromal subunits. A first model of PsaD consisting of a four-stranded beta-sheet and an alpha-helix is suggested, indicating that this subunit partly shields PsaC from the stromal side. In addition to the improvements on the stromal subunits, the structural model of the membrane-integral region of PSI is also extended. The current electron density map allows the identification of the N and C termini of the subunits PsaA and PsaB. The 11-transmembrane alpha-helices of these subunits can now be assigned uniquely to the hydrophobic segments identified by hydrophobicity analyses.

Localization of two phylloquinones, QK and QK', in an improved electron density map of photosystem I at 4-A resolution.

An improved electron density map of photosystem I from Synechococcus elongatus calculated at 4-A resolution for the first time reveals a second phylloquinone molecule and thereby completes the set of cofactors constituting the electron transfer system of this iron-sulfur type photosynthetic reaction center: six chlorophyll a, two phylloquinones, and three Fe4S4 clusters. The location of the newly identified phylloquinone pair, the individual plane orientations of these molecules, and the resulting distances to other cofactors of the electron transfer system are discussed and compared with those determined by magnetic resonance techniques.

A common ancestor for oxygenic and anoxygenic photosynthetic systems: a comparison based on the structural model of photosystem I.

The 4 A structural model of photosystem I (PSI) has elucidated essential features of this protein complex. Inter alia, it demonstrates that the core proteins of PSI, PsaA and PsaB each consist of an N-terminal antenna-binding domain, and a C-terminal reaction center (RC)-domain. A comparison of the RC-domain of PSI and the photosynthetic RC of purple bacteria (PbRC), reveals significantly analogous structures. This provides the structural support for the hypothesis that the two RC-types (I and II) share a common evolutionary origin. Apart from a similar set of constituent cofactors of the electron transfer system, the analogous features include a comparable cofactor arrangement and a corresponding secondary structure motif of the RC-cores. Despite these analogies, significant differences are evident, particularly as regards the distances between and the orientation of individual cofactors, and the length and orientation of alpha-helices. Inferred roles of conserved amino acids are discussed for PSI, photosystem II (PSII), photosystem C (PSC, green sulfur bacteria) and photosystem H (PSH, heliobacteria). Significant sequence homology between the N-terminal, antenna-binding domains of the core proteins of type-I RCs, PsaA, PsaB, PscA and PshA (of PSI, PSC and PSH respectively) with the antenna-binding subunits CP43 and CP47 of PSII indicate that PSII has a modular structure comparable to that of PSI.

Photosystem I of Synechococcus elongatus at 4 A resolution: comprehensive structure analysis.

An improved structural model of the photosystem I complex from the thermophilic cyanobacterium Synechococcus elongatus is described at 4 A resolution. This represents the most complete model of a photosystem presently available, uniting both a photosynthetic reaction centre domain and a core antenna system. Most constituent elements of the electron transfer system have been located and their relative centre-to-centre distances determined at an accuracy of approximately 1 A. These include three pseudosymmetric pairs of Chla and three iron-sulphur centres, FX, FA and FB. The first pair, a Chla dimer, has been assigned to the primary electron donor P700. One or both Chla of the second pair, eC2 and eC'2, presumably functionally link P700 to the corresponding Chla of the third pair, eC3 and eC'3, which is assumed to constitute the spectroscopically-identified primary electron acceptor(s), A0, of PSI. A likely location of the subsequent phylloquinone electron acceptor, QK, in relation to the properties of the spectroscopically identified electron acceptor A1 is discussed. The positions of a total of 89 Chla, 83 of which constitute the core antenna system, are presented. The maximal centre-to-centre distance between antenna Chla is < or = 16 A; 81 Chla are grouped into four clusters comprising 21, 23, 17 and 20 Chla, respectively. Two "connecting" Chla are positioned to structurally (and possibly functionally) link the Chla of the core antenna to those of the electron transfer system. Thus the second and third Chla pairs of the electron transfer system may have a dual function both in energy transfer and electron transport. A total of 34 transmembrane and nine surface alpha-helices have been identified and assigned to the 11 subunits of the PSI complex. The connectivity of the nine C-terminal (seven transmembrane, two "surface") alpha-helices of each of the large core subunits PsaA and PsaB is described. The assignment of the amino acid sequence to the transmembrane alpha-helices is proposed and likely residues involved in co-ordinating the Chla of the electron transfer system discussed.

Pulsed EPR structure analysis of photosystem I single crystals: localization of the phylloquinone acceptor.

A novel application of electron paramagnetic resonance (EPR) is reported to gain three dimensional structural information on cofactors in proteins. The method is applied here to determine the unknown position of the electron acceptor QK, a phylloquinone (vitamin K1), in the electron transfer chain in photosystem I of oxygenic photosynthesis. The unusual electron spin echo (out-of-phase echo) observed for the light induced radical pair P700.+QK.- in PS I allows the measurement of the dipolar coupling between the two radical pair spins which yields directly the distance between these two radicals. Full advantage of the information in the out-of-phase echo modulation can be taken if measurements using single crystals are performed. With such samples, the orientation of the principal axis of the dipolar interaction, i.e., the axis connecting P700.+QK.-, can be determined with respect to the crystal axes system. An angle of theta = (27 +/- 5)degrees between the dipolar coupling axis and the crystallographic c-axis has been derived from the modulation of the out-of-phase echo. Furthermore, the projection of the dipolar axis into the crystallographic a,b-plane, is found to be parallel to the a-axis. The results allow for the determination of two possible locations of QK within the electron transfer chain of photosystem I. These two positions are related to each other by the pseudo C2 symmetry of the chlorophyll cofactors.

Photosystem I at 4 A resolution represents the first structural model of a joint photosynthetic reaction centre and core antenna system.

The 4 A X-ray structure model of trimeric photosystem I of the cyanobacterium Synechococcus elongatus reveals 31 transmembrane, nine surface and three stromal alpha-helices per monomer, assigned to the 11 protein subunits: PsaA and PsaB are related by a pseudo two-fold axis normal to the membrane plane, along which the electron transfer pigments are arranged. 65 antenna chlorophyll a (Chl a) molecules separated by < or = 16 A form an oval, clustered net continuous with the electron transfer chain through the second and third Chl a pairs of the electron transfer system. This suggests a dual role for these Chl a both in excitation energy and electron transfer. The architecture of the protein core indicates quinone and iron-sulphur type reaction centres to have a common ancestor.

Functional mechanism of water splitting photosynthesis.

A personal account is given on physico-chemical aspects of photosynthesis. The article starts with the way I entered the field of photosynthesis. Then, selected results from our research group are discussed. Three methods used for functional analysis in our laboratory are described: the repetitive flash spectroscopy; the electrochromic volt- and ammeter; and the membrane energization by a battery. Our subsequent studies deal with the two photoreaction centers, the primary charge separation, the plastoquinones as a transmembrane link between the two centers and the vectorial electron- and proton pathways. The results led to a picture of the elementary functional mechanism of the molecular machinery in the thylakoid membrane. The perspective then focuses on the coupling between the electric field, protons and phosphorylation. This section is followed by our observations and analysis of the mechanism of water cleavage and its coupling with the functioning of reaction center II. Finally, information is provided on structural aspects of the two reaction centers. The article ends with a retrospect.

Reaction sequences from light absorption to the cleavage of water in photosynthesis : Routes, rates and intermediates.

The reaction sequence between the primary electron acceptor, the oxidized Chlorophyll-aII, and the terminal electron donor, the water splitting enzyme system S, is being described in the range from nanoseconds to milliseconds. For the cleavage of water Chlorophyll-aII (+) extracts four electrons in four turnovers from the enzyme system S responsible for the water oxidation. For each extraction the electron is moved step by step along the chain that connects the Chlorophyll-aII center with that of S. Beginning with the transfer from the immediate donor, D1, to Chl-aII (+), the subsequent transfer from D2 to D1 (+) ends in the electron transfer from S to D2 (+). This final act establishes in S the oxidizing equivalent, probably in the form of oxidized manganese. Coupled with these acts is an intrinsic proton release and a surplus charge formation. After the generation of the 4th oxidizing equivalent in a concerted final action the evolution of O2 from water takes place. Correlations between the events are described quantitatively.

Total recovery of O2 evolution and nanosecond reduction kinetics of chlorophyll-a II (+) (P-680 (+)) after inhibition of water cleavage with acetate.

Oxygen evolution and reduction kinetics of the photooxidized Chl-aII (+) have been measured in oxygen-evolving complexes from the thermophilic cyanobacterium Synechococcus sp. 1. Incubation of PS II particles with acetate resulted in an inhibition of oxygen evolution and a retardation of the Chl-aII (+)=reduction kinetics from the nanosecond range to the microsecond range, indicating a modification of the donor side of photosystem II (PS II). 2. After the first two flashes given to a dark-adapted, acetate treated sample, Chl-aII (+) was re-reduced with a half-life time of 160 µs by a component of the donor side of PS II. Under repetitive excitation Chl-aII (+) was re-reduced in 500 µs by electron back reaction from the primary acceptor QA (-) (X-320(-)). Obviously, in the presence of acetate only two electrons are available from the donor side. 3. Both oxygen evolution and nanosecond reduction kinetics of Chl-aII (+) were restored to the control level when acetate was removed. 4. The results indicate a tight coupling between O2 evolution and nanosecond reduction kinetics of Chl-aII (+). 5. The reversible inhibition is probably due to a replacement of Cl(-) by acetate within the water splitting enzyme. 6. Due to its strongly retarded kinetics, the reversibly modified system may facilitate investigations of the mechanism of the donor side.

Relation between the initial kinetics of ATP synthesis and of conformational changes in the chloroplast ATPase studied by external field pulses.

ATP formation and the energy-dependent release of tightly bound [14C]-adenine nucleotides from the chloroplast coupling factor CF1 has been studied as a function of the time of energization of the membrane in the range of 500 mus up to 60 ms. The high time resolution was achieved because the energization was generated artificially by external electric field pulses. Applying external electric field pulses to a chloroplast suspension induces an electric potential difference across the thylakoid membrane. The following results were obtained: (1) The amount of ATP generated increases linearly with the time of energization. The steady-state rate of ATP formation is reached in less than 500 mus. (2) A fraction of the adenine nucleotides tightly bound to CF1 is released on energization with a half-rise-time of about 2 ms. The size of the fraction, i.e., the amplitude of the fast phase of the release, increases with the magnitude of the induced transmembrane electric potential difference. A further slow release is superimposed. (3) The initial rate of the release of adenine nucleotides is practically identical with the rate of ATP formation. It is concluded that the release of tightly bound nucleotides monitors an initial conformational change by which the ATPase turns from an inactive into an activated state. For the explanation of the results a reaction scheme is proposed which takes into account a preceding activation of the ATPase.

The plastoquinone pool as possible hydrogen pump in photosynthesis.

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H+ + e-) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H+ uptake and the internal H+ release led to the following conclusions: (1) A stoichiometric coupling of 1 : 1 : 1 between the external H+ uptake, the electron translocation through the plastoquinone pool and the internal H+ release (corrected for H+ release due to H2O oxidation) is valid (pHout = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation. These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H+ + e-) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.

Effect of electric fields on the absorption spectrum of dye molecules in lipid layers. V. Refined analysis of the field-indicating absorption changes in photosynthetic membranes by comparison with electrochromic measurements in vitro.

The comparison of light-induced absorption changes in photosynthesis with electrochromic spectra of the isolated pigments in vitro is renewed more thoroughly and described in detail, involving new measurements of the linear electrochromism of oriented chlorophyll beta [6]. 1. The coincidence of the maxima and minima in the in vivo spectrum with those in the in vitro superposition is better than in previous studies [4]. 2. The molar ratio of the pigments now used for the superposition of the in vitro spectra is the same as that in vivo. 3. From this and from surface-pressure/area diagrams of the chlorophylls on a water surface, conclusions are drawn concerning the preferential orientations of the dipole moment differences of the red and blue absorption bands of the bulk chlorophylls in the membrane. 4. From the comparison of the electrochromism of the carotenoids with the absorption change at 520 nm in vivo, it is concluded that the bulk of the carotenoids are oriented at a rather flat angle in the membrane (approximately 16 degrees).