The structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225Å and a diameter of approximately 100Å. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300Å (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28Å only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225Å can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28Å. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively.

In adaption to its specific environmental conditions, the cyanobacterium Acaryochloris marina developed two different types of light-harvesting complexes: chlorophyll-d-containing membrane-intrinsic complexes and phycocyanobilin (PCB) - containing phycobiliprotein (PBP) complexes. The latter complexes are believed to form a rod-shaped structure comprising three homo-hexamers of phycocyanin (PC), one hetero-hexamer of phycocyanin and allophycocyanin (APC) and probably a linker protein connecting the PBPs to the reaction centre. Excitation energy transfer and electron-vibrational coupling in PBPs have been investigated by selectively excited fluorescence spectra. The data reveal a rich spectral substructure with a total of five low-energy electronic states with fluorescence bands at 635nm, 645nm, 654nm, 659nm and a terminal emitter at about 673 nm. The electronic states at ~635 and 645 nm are tentatively attributed to PC and APC, respectively, while an apparent heterogeneity among PC subunits may also play a role. The other fluorescence bands may be associated with three different isoforms of the linker protein. Furthermore, a large number of vibrational features can be identified for each electronic state with intense phonon sidebands peaking at about 31 to 37cm⁻¹, which are among the highest phonon frequencies observed for photosynthetic antenna complexes. The corresponding Huang-Rhys factors S fall in the range between 0.98 (terminal emitter), 1.15 (APC), and 1.42 (PC). Two characteristic vibronic lines at about 1580 and 1634cm⁻¹ appear to reflect CNH⁺ and CC stretching modes of the PCB chromophore, respectively. The exact phonon and vibrational frequencies vary with electronic state implying that the respective PCB chromophores are bound to different protein environments. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

We Investigated The Excitation Energy Transfer (Eet) In The Phycobiliprotein (Pbp) Antenna Of The Chl D-Containing Cyanobacterium Acaryochloris Marina. The Equilibration Of The Excitation Energy Over All Phycocyanin (Pc) And Allophycocyanin (Apc) Molecules Of The Pbpantenna In A. Marina Was Found To Occur With Time Constants Of 400 Fs, 3 Ps And 14 Ps. This Is More Than Ten Times Faster Than The Eet From The Pc Rod-Antenna To The Apc Core In Typical Cyanobacteria With Phycobilisomes As Synechococcus 6301 (Holzwarth 1991). These Results Are In Agreement With The Unique Structure Of The Pbp-Antenna Of A. Marina Which Consists Of Three Pc-Homohexamers And One Hetero-Hexamer Containing Pc And Apc (Marquardt Et Al. 1997). The Results Suggest That The Presence Of Apc And Pc Within One Hexamer, Which Has So Far Been Observed Only In A. Marina, Enables A Very Fast 3 Ps Eet From Pc To Apc, Thereby Facilitating A Fast Energy Transfer From Phycobiliproteins To Ps Ii With A Time Constant Of 70 Ps (Petrasek Et Al. 2005).

The transients of normalized fluorescence yield induced by an actinic laser flash in dark adapted leaves of Arabidopsis thaliana plants were measured with new equipment, that was developed as part of this work and permits the covarage of a wide time domain of 8 decades from 100 ns to 10 s. The raw data obtained were processed and analyzed within the framework of the "3-quencher" model with Q(A) as photochemical and P680(+)(*) and (3)Car as nonphotochemical quenchers. Comparative measurements with hydroxylamine treated PS II membrane fragments from spinach revealed that the widely used "dogma"of virtually identical efficiency of photochemical (Q(A)) and nonphotochemical (P680(+)(*)) quenching has to be revised: the constant of the latter exceeds that of the former by a factor of about 2. As a consequence, the probability of recombination between P680(+)(*) and Q(A)(-) and its kinetics have to be explicitly taken into account for the interpretation of flash induced fluorescence yield transients. The analysis of the experimental data within this extended "3-quencher" model reveals that a fully consistent description is achieved for the data gathered from measurements with intact leaves from wild type plants excited with actinic laser flashes of different energies (number of photons per flash and unit area). On the basis of these results it is shown that, in dark adapted leaves excited with a single laser flash, P680(+)(*) is predominantly (about 80% of the total reaction) reduced by Y(Z) via nanosecond kinetics and Q(A)(-) reoxidation is dominated by a kinetics of about 150 mus that are ascribed to PS II complexes with the Q(B) site occupied by PQ. The excess of excited chlorophyll singlet states decays to a significant extent via the carotenoid "triplet valve"with transient population of (3)Car. The present data provide the basis for analyses of A. thaliana mutants with modified lipid content and composition. The results of these investigations are described in an accompanying report (Steffen, R., Kelly, A. A., Huyer, J., Dormann, P., and Renger, G. (2005) Investigations on the reaction pattern of photosystem II in leaves from Arabidopsis thaliana wild type plants and mutants with genetically modified lipid content, Biochemistry 44, 3134-3142).

The multiphasic P680(+.) reduction kinetics by Y-Z and their temperature dependence were investigated in PS II core complexes with high oxygen evolution capacity, isolated from a thermophilic cyanobacterium (Thermosynechococcus elongatus) and a higher plant (Spinacea oleracea). Measurements and kinetic analyses of laser flash induced 820 nm absorption changes (reflecting the turnover of P680) led to the following results: (a) the pattern of multiphasic P680(+.) reduction is basically the same in both species, (b) the activation energy of the "fast" nanosecond kinetics is 20 +/- 5 kJ mol(-1) and 14 +/- 5 kJ mol(-1) for the samples from T. elongatus and S. oleracea, respectively, (c) the activation energies of this reaction are nearly the same in complexes with water oxidizing complex (WOC) in redox states S-1 and S-2, (d) the activation energy of the "slow" nanosecond kinetics ascribed to "local" relaxation processes is larger by almost a factor of two compared to that of the "fast" nanosecond kinetics, and (e) the normalized amplitudes of the "fast" and "slow" nanosecond kinetics are virtually independent of temperature in the physiological range for PS II core complexes from both organisms. Based on these findings the energetics and kinetics of P680(+.) reduction in fully competent PS II is briefly discussed within the framework of a dynamic model of sequential relaxation processes. The protein dynamics are inferred to provide the major contribution to the driving force of the redox reaction.

The decay kinetics of chlorophyll (Chl) fluorescence of solubilized pigment protein complexes of the distal, proximal, and core antenna of photosystem II from higher plants have been analyzed in the temperature range of 10-277 K using buffer solutions containing or lacking sucrose as cryoprotectant. It was found that (i) at 277 K the (1)Chl* decay of the complexes LHCIIb (distal), CP29 (proximal), and CP47 (core) is characterized by a biphasic kinetics with characteristic lifetimes in the range of 1.5-2.5 ns (fast phase) and 4-4.8 ns (slow phase), (ii) the slow phase dominates in all three complexes with normalized amplitudes of greater than or equal to65, (iii) in solutions containing sucrose the lifetime of the slow phase increases with decreasing temperature and reaches values in the range of 5.2-5.8 ns at 10 K, whereas those of the fast phase exhibit a more complex temperature dependence with a pronounced minimum value in the range of 150-200 K, (iv) markedly different temperature dependencies with pronounced minima in the range of 150-200 K are observed for both lifetime and normalized amplitude of the slow phase when the pigment protein complexes are dissolved in buffers without sucrose. The results are interpreted as evidence for two spectroscopically and kinetically distinguishable subpopulations in solubilized LHCIIb, CP29, and CP47 that are characterized by different rate constants of radiationless decay into the ground state of Chl. Possible mechanisms are discussed.

The adverse effect of low intensity, small band UV-B irradiation (λ = 305 ± 5 nm, I = 300 mW m −2 ) on PS II has been studied by comparative measurements of laser flash-induced changes of the absorption at 325 nm, ΔA 325 (t), as an indicator of redox changes in Q A , and of the relative fluorescence quantum yield, F(t)/F o , in PS II membrane fragments. The properties of untreated control were compared with those of samples where the oxygen evolution rate under illumination with continuous saturating light was inhibited by up to 95%. The following results were obtained: a) the detectable initial amplitude (at a time resolution of 30 μs) of the 325 nm absorption changes, ΔA 325 , remained virtually invariant whereas the relaxation kinetics exhibit significant changes, b) the 300 μs kinetics of ΔA 325 dominating the relaxation in UV-B treated samples was largely replaced by a 1.3 ms kinetics after addition of MnCl 2 , c) the extent of the flash induced rise of the relative fluorescence quantum yield was severely diminished in UV-B treated PS II membrane fragments but the relaxation kinetics remain virtually unaffected. Based on these results the water oxidizing complex (WOC) is inferred to be the primary target of UV-B impairment of PS II while the formation of the ‘stable’ radical pair P680 +· Q A −● is almost invariant to this UV-B treatment.

Photosynthetic oxidation of two water molecules to molecular oxygen and four protons comprises three types of reactions (for a review, see Renger 1999): i) generation of a strongly oxidizing Chlorophyll-a cation radical (P680) by light induced charge separation, ii) transfer of this oxidizing redox equivalent to tyrosine residue YZ under formation of the neutral radical YZ and iii) stepwise electron abstraction by YZ from the water oxidizing complex (WOC). In addition to the redox active tetranuclear manganese cluster a single Ca-ion is indispensable for the functional competence of the WOC (for a review, see Debus 1992). Different treatments were used to remove Ca in order to study restoration of oxygen evolution capacity and rebinding of Ca (Ghanotakis et al. 1984, Adelroth et al. 1995). A surprising heterogeneity of restoration and/or binding constants was found (Kalosaka et al. 1990, Adelroth et al. 1995) that rises questions on the origin of this phenomenon. It could reflect either a heterogeneity of WOCs or multiple effects of Ca or a combination of both. In a recent report Ca was inferred to be not only an essential constituent of the WOC but also to exert an important regulatory function in the mode of coupling between membrane energization and ATP-synthesis at limiting light intensities in relation to switching on nonphotochemical quenching under light stress. It was postulated that Ca binding to the CF0 part of ATP-ase exerts the regulatory control (Pan and Dilley 2000). The present communication describes effects of low affinity Ca binding and thermal activation on the multiphasic kinetics of P680 reduction by YZ in solubilized untreated spinach PS II core complexes with high oxygen evolving capacity.

The temperature dependence of donor side reactions was analysed within the framework of the Marcus theory of nonadiabatic electron transfer. The following results were obtained for PS II membrane fragments from spinach: (1) the reorganisation energy of P680+• reduction by YZ is of the order of 0.5 eV in samples with a functionally fully competent water oxidising complex (WOC); (2) destruction of the WOC by Tris-washing gives rise to a drastic increase of λ to values of the order of 1.6 eV; (3) the reorganisation energies of the oxidation steps in the WOC are dependent, on the redox states S i with values of about 0.6 eV for the reactions YZOXS0→YZS1 and YZOXS1→YZS2, 1.6 eV for the reaction YZOXS2→YZS3 and 1.1 eV (above a characteristic temperature uc of about 6  °C) for the reaction YZOXS3→→YZS0+O2. Using an empirical rate constant-distance relationship, the van der Waals distance between YZ and P680 was found to be about 10 Å, independent of the presence or absence of the WOC, whereas the distance between YZ and the manganese cluster in the WOC was ≥15 Å. Based on the calculated activation energies the environment of YZ is inferred to be almost "dry" and hydrophobic when the WOC is intact but becomes enriched with water molecules after WOC destruction. Furthermore, it is concluded that the transition S2→S3 is an electron transfer reaction gated by a conformational change, i.e. it comprises significant structural changes of functional relevance. Measurements of kinetic H/D isotope exchange effects support the idea that none of these reactions is gated by the break of a covalent O-H bond. The implications of these findings for the mechanism of water oxidation are discussed.

Out-of-phase electron spin echo envelope modulation (ESEEM) spectroscopy was used to determine the distance between the primary donor radical cation P680+. and the quinone acceptor radical anion Q(A)-. in iron-depleted photosystem II in membrane fragments from spinach that are deprived of the water oxidizing complex. Furthermore, a lower limit for the distance between the oxidized tyrosine residue Y(Z) of polypeptide D1 and Q(A)-. could be estimated by a comparison of data gathered from samples where the electron transfer from Y(Z) to P680+. is either intact or blocked by preillumination in the presence of NH2OH.

The influence of H/D-exchange on the electron transfer from Y-Z to P680(+.), and the recombination reaction between P680(+.) and Q(A)(-.) in Tris-treated photosystem 2 (PS2) membrane fragments at pL [L=lyonium ion (H,D)] = 6.5 was investigated by monitoring and numerical analysis of flash-induced absorption changes at 830 and 320 nm, respectively. The H/D-exchange caused retardation by a factor of approximately 3 of the electron transfer from Y-Z to P680(+.). In marked contrast, no significant effect was observed on the kinetics of P680(+.)Q(A)(-.) charge recombination. In addition, the pH-dependence of P680(+.)Q(A)(-.) recombination kinetics were analysed in samples where Y-Z was functionally eliminated by exposure of Tris-treated PS2 fragments to strong irradiance. In this case the relaxation kinetics could be fitted by three-exponentials with half lifetimes of 150 mu s (fast), 800 mu s (middle) and 10 ms (slow) at pH = 6.0. The fast and middle kinetics were only slightly dependent on pH in the range from 5.0 to 8.0. On the other hand, the normalised amplitudes of these kinetics were markedly pH-dependent. Furthermore, the normalised extent of the slow kinetics was significantly larger in the absorption changes at 320 nm, reflecting the turnover of Q(A), than at 830 nm as an indicator of P680(+.) formation and decay. One possible explanation of this feature is provided by an assumption that Tris-washed PS2 membrane fragments exposed to a strong irradiance contain a redox component competing with Q(A)(-.) in the reduction of P680(+.). Furthermore, the pH-dependent changes of the overall kinetics of P680(+.)Q(A)(-.) recombination originated predominantly from different ratios of the extent of the fast and middle components rather than from marked modifications of the rate constants.

Chlorophyll a content and maximum yield of radical ion pair formation, P680 +· Pheo -· , were analyzed in D1/D2/cyt b 559 preparations isolated from spinach. The following results were obtained: (a) chromatographic analysis with HPLC using an area-ratio method independent of extinction coefficients revealed that 7±0.5 Chl a are present per 2 Pheo; (b) based on highly sensitive silver staining of proteins and its densitometric evaluation, the contamination by CP47 was estimated to be 4%, i.e., no more than 0.8 Chl can be ascribed to this Chl a binding protein; (c) measurements of laser flash induced absorption changes at 830 nm as a function of the pulse energy indicate that under light saturating conditions one P680 +· Pheo −· radical pair can be formed per 7–8 Chl a ; (d) based on the saturation behavior of the different decay components of the absorption changes and on a comparison of the absorption and single photon counting fluorescence data, the 5-ns component is inferred to originate from `disconnected' Chl. These results lead to the conclusion that functionally fully competent D1/D2/cyt b 559 complexes contain 6 Chl per 2 Pheo. More than 90% of the centres are able to perform a charge separation leading to the radical pair P680 +· Pheo −· .

H/D isotope exchange effects on P680+· reduction by Yz and electron abstraction from the water oxidizing complex (WOC) in redox state S3 by YZOX were analyzed in PS II core complexes from spinach by measurements of laser flash induced absorption changes at 820 nm and 355 nm. The results obtained reveal: (1) the rate of YZ oxidation by P680+· is almost independent of the substitution of exchangeable protons by deuterons; and (2) the reaction between YZOX and the WOC in S3 exhibits a kinetic H/D isotope exchange effect of similar magnitude as that recently observed in PS II membrane fragments [Renger, G., Bittner, T. and Messinger, J. (1994) Biochem. Soc. Trans. 22, 318–322]. Based on these results it is inferred that photosynthetic dioxygen formation comprises the cleavage of at least one hydrogen bond.

Measurements of time-resolved fluorescence decay, laser-flash-induced absorption changes in the UV and at 820 nm and of the relative fluorescence quantum yield in different preparations (thylakoids, photosystem II (PSII) membrane fragments and PSII core complexes) from spinach led to a number of conclusions. (1) Light is transformed into Gibbs energy with trapping times of 250 ps and 130 ps in open reaction centres of PSII membrane fragments and PSII core complexes, respectively. Assuming rapid Boltzmann distribution of excitation energy and taking into account the antenna properties (size and spectral distribution), the molecular rate constant of primary charge separation is estimated to be about (3 ps)-1. (2) The electron transfer from Pheo- to QA is characterised by a rate constant of (300 ps)-1. (3) The QA- reoxidation kinetics are significantly retarded in D2O suspensions. These H/D isotope effects are interpreted as to reflect hydrogen-bond dependent changes in the protein dynamics that are relevant to electron transfer. (4) In PSII reaction centres closed for photochemical trapping the yield of a primary radical pair with lifetimes exceeding 1 ns is comparatively small (c 30%) at room temperature. Short illumination in the presence of Na2S2O4 changes the radical pair dynamics. (5) Photoinhibition under aerobic conditions impairs the primary charge separation and leads to formation of quencher(s) of excitation energy.

Measurements of flash-induced absorption changes at 325, 436, and 830 nm and of oxygen evolution were performed in order to analyze in detail the inhibition of photosystem II (PS II) by Cu(II) in PS II membrane fragments from spinach. (a) The kinetics of P680+ reduction become markedly slower in the presence of 100 microM CuSO4. (b) The CuSO4-induced kinetics of P680+ reduction are dominated by a 140-160-microsecond decay. (c) The extent of these 140-160-microsecond kinetics, normalized to the overall decay, remains virtually unaffected by addition of the exogenous PS II donor, NH2OH. (d) In thoroughly dark-adapted samples the CuSO4-induced 140-160-microsecond kinetics are already observed after the first flash and remain unchanged by a train of excitation flashes. (e) The extent of P680+ and QA- formation under repetitive flash excitation is not diminished by addition of 100 microM CuSO4. (f) The induction of microsecond kinetics of P680+ reduction at the expense of ns kinetics and the inhibition of the saturation rate of oxygen evolution exhibit the same dependence on CuSO4 concentration. (g) CuSO4 also transforms the 10-20-microsecond reduction of P680+ by TyrZ in Tris-washed PS II membrane fragments into 140-160-microsecond kinetics without any effect on the extent of flash-induced P680+ formation. These results unambiguously show that Cu(II) does not affect the charge separation (P680+QA-), but instead specifically modifies TyrZ and/or its micro environment so that the electron transfer to P680+ becomes blocked.

The stabilization of the primary radical pair P680(+) pheophytin (Pheo)(-) through rapid electron transfer from Pheo(-) to the special plastoquinone of photosystem II (PS II), Q(A), was analyzed on the basis of time-resolved (40 ps) UV-absorption changes detected in different PS II preparations from higher plants. Lifetime measurements of (1)Chl* fluorescence by single photon counting and a numerical analysis of the redox reactions revealed (1) at exciton densities required for light saturation of the stable charge separation, annihilation processes dominate the excited state decay leading to very similar lifetimes of (1)Chl* in systems with open and closed reaction centers and (2) the difference of absorption changes induced by actinic flashes of comparatively high photon density in samples with open and photochemically closed reaction centers, respectively, provides a suitable measure of the rate constant of Q(A)(-) formation. Conclusion 2 was confirmed in PS II membrane fragments by measurements at three wavelengths (280 nm, 292 nm and 325 nm) where the difference spectrum of Q(A)(-) formation exhibits characteristic features. The numerical evaluation of the experimental data led to the following results: (1) the rate constant of Q(A)(-) formation was found to be (300 +/- 100 ps)(-1) in PS II membrane fragments and PS II core complexes deprived of the distal and proximal antenna and (2) an iron depletion treatment of membrane fragments does not affect these kinetics. The implications of these results are briefly discussed in terms of the PS II reaction pattern.

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy γ-rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the QB site: H2O → (Mn)4 → Yz → P680 → Pheophytin → Q → phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the α and β subunits of cytochrome b 559and the 4.5 kDa psbI gene product.

Yield and decay kinetics of the laser flash-induced primary radical pair P680+Pheo− were analyzed by measuring flash-induced absorption changes at 820 nm (ΔA820) and fluorescence decay kinetics in PS II core complexes under different redox conditions for the primary plastoquinone acceptor, QA. If QA is chemically reduced in the dark by Na2S2O4, the yield of long-lived (τ > 1 ns) P680+Pheo− states is 30% with lifetimes of 4 ns (about 23 of the total decay) and 30 ns (about 13 of the total decay). The fluorescence data suggest that the 30 ns radical pair and probably the 4 ns radical pair as well decay predominately by recombination to P680∗Pheo. Irradiation of PS II core complexes in the presence of Na2S2O4 for only 10 s with intense visible light increases the yield of the 30 ns primary radical pair by a factor of 3. This result can be explained by a double reduction of QA and subsequent protonation leading to QAH2. At short irradiation times this effect is reversible because subsequent addition of K3[Fe(CN)6] fully restores the capability for a stable charge separation giving rise to P680+QA formation. However, longer irradiation of Na2S2O4-treated PS II core complexes leads to an irreversible impairment of the primary charge separation.

Primary events in the photoinactivation of photosystem (PS) 2 membrane fragments by low and high ''visible light'' irradiance (17 to 1700 W m-2) and UV-B irradiation (90 W m-2) were analyzed by measuring flash-induced absorption changes at 830 nm that reflect transient formation of P680+ and Pheo-. Following results were obtained: (1) Irradiation with ''visible light'' under aerobic conditions affects the PS 2 electron transfer at two different sites: (a) within the PS 2 reaction centre by impairment of primary charge separation (P680 Pheo Q(A) --> P680+Pheo-Q(A)), and (b) on the PS 2 donor side by inhibition of the electron transfer from Y(Z) to P680+. (2) In PS 2 membrane fragments with intact O2-evolution the primary charge separation is the most sensitive target of the photoinhibition by ''visible light''. The UV-B irradiation, however, affects predominantly the oxygen-evolving complex or the electron transfer from the oxygen-evolving complex to Y(Z)ox. (3) Susceptibility of the P680 Y(Z) segment to photoinhibition by ''visible light'' is drastically increased in the samples with lifetimes of Y(Z)ox and P680+ having been significantly prolonged by Tris-treatment. Susceptibility of the primary charge separation to photoinhibition, however, is not dependent on the lifetimes of P680+ and Y(Z)ox.