Eberhard Schlodder

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).

Psb27, a Cyanobacterial Lipoprotein, Is Involved in the Repair Cycle of Photosystem II

Photosystem II (PSII) performs one of the key reactions on our planet: the light-driven oxidation of water. This fundamental but very complex process requires PSII to act in a highly coordinated fashion. Despite detailed structural information on the fully assembled PSII complex, the dynamic aspects of formation, processing, turnover, and degradation of PSII with at least 19 subunits and various cofactors are still not fully understood. Transient complexes are especially difficult to characterize due to low abundance, potential heterogeneity, and instability. Here, we show that Psb27 is involved in the assembly of the water-splitting site of PSII and in the turnover of the complex. Psb27 is a bacterial lipoprotein with a specific lipid modification as shown by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The combination of HPLC purification of four different PSII subcomplexes and 15N pulse label experiments revealed that lipoprotein Psb27 is part of a preassembled PSII subcomplex that represents a distinct intermediate in the repair cycle of PSII.

Time-Resolved Fluorescence Emission Measurements of Photosystem I Particles of Various Cyanobacteria: A Unified Compartmental Model

Photosystem I (PS-I) contains a small fraction of chlorophylls (Chls) that absorb at wavelengths longer than the primary electron donor P700. The total number of these long wavelength Chls and their spectral distribution are strongly species dependent. In this contribution we present room temperature time-resolved fluorescence data of five PS-I core complexes that contain different amounts of these long wavelength Chls, i.e., monomeric and trimeric photosystem I particles of the cyanobacteria Synechocystis sp. PCC 6803, Synechococcus elongatus, and Spirulina platensis, which were obtained using a synchroscan streak camera. Global analysis of the data reveals considerable differences between the equilibration components (3.4-15 ps) and trapping components (23-50 ps) of the various PS-I complexes. We show that a relatively simple compartmental model can be used to reproduce all of the observed kinetics and demonstrate that the large kinetic differences are purely the result of differences in the long wavelength Chl content. This procedure not only offers rate constants of energy transfer between and of trapping from the compartments, but also well-defined room temperature emission spectra of the individual Chl pools. A pool of red shifted Chls absorbing around 702 nm and emitting around 712 nm was found to be a common feature of all studied PS-I particles. These red shifted Chls were found to be located neither very close to P700 nor very remote from P700. In Synechococcus trimeric and Spirulina monomeric PS-I cores, a second pool of red Chls was present which absorbs around 708 nm, and emits around 721 nm. In Spirulina trimeric PS-I cores an even more red shifted second pool of red Chls was found, absorbing around 715 nm and emitting at 730 nm.

Light Harvesting in Photosystem I: Modeling Based on the 2.5-Å Structure of Photosystem I from Synechococcus elongatus

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus has been recently resolved by x-ray crystallography to 2.5-A resolution. Besides the reaction center, photosystem I consists also of a core antenna containing 90 chlorophyll and 22 carotenoid molecules. It is their function to harvest solar energy and to transfer this energy to the reaction center (RC) where the excitation energy is converted into a charge separated state. Methods of steady-state optical spectroscopy such as absorption, linear, and circular dichroism have been applied to obtain information on the spectral properties of the complex, whereas transient absorption and fluorescence studies reported in the literature provide information on the dynamics of the excitation energy transfer. On the basis of the structure, the spectral properties and the energy transfer kinetics are simultaneously modeled by application of excitonic coupling theory to reveal relationships between structure and function. A spectral assignment of the 96 chlorophylls is suggested that allows us to reproduce both optical spectra and transfer and emission spectra and lifetimes of the photosystem I complex from S. elongatus. The model calculation allowed to study the influence of the following parameters on the excited state dynamics: the orientation factor, the heterogeneous site energies, the modifications arising from excitonic coupling (redistribution of oscillator strength, energetic splitting, reorientation of transition dipoles), and presence or absence of the linker cluster chlorophylls between antenna and reaction center. For the Förster radius and the intrinsic primary charge separation rate, the following values have been obtained: R(0) = 7.8 nm and k(CS) = 0.9 ps(-1). Variations of these parameters indicate that the excited state dynamics is neither pure trap limited, nor pure transfer (to-the-trap) limited but seems to be rather balanced.

Energy Transfer and Charge Separation in Photosystem I: P700 Oxidation Upon Selective Excitation of the Long-Wavelength Antenna Chlorophylls of Synechococcus elongatus

Photosystem I of the cyanobacterium Synechococcus elongatus contains two spectral pools of chlorophylls called C-708 and C-719 that absorb at longer wavelengths than the primary electron donor P700. We investigated the relative quantum yields of photochemical charge separation and fluorescence as a function of excitation wavelength and temperature in trimeric and monomeric photosystem I complexes of this cyanobacterium. The monomeric complexes are characterized by a reduced content of the C-719 spectral form. At room temperature, an analysis of the wavelength dependence of P700 oxidation indicated that all absorbed light, even of wavelengths of up to 750 nm, has the same probability of resulting in a stable P700 photooxidation. Upon cooling from 295 K to 5 K, the nonselectively excited steady-state emission increased by 11- and 16-fold in the trimeric and monomeric complexes, respectively, whereas the quantum yield of P700 oxidation decreased 2.2- and 1.7-fold. Fluorescence excitation spectra at 5 K indicate that the fluorescence quantum yield further increases upon scanning of the excitation wavelength from 690 nm to 710 nm, whereas the quantum yield of P700 oxidation decreases significantly upon excitation at wavelengths longer than 700 nm. Based on these findings, we conclude that at 5 K the excited state is not equilibrated over the antenna before charge separation occurs, and that approximately 50% of the excitations reach P700 before they become irreversibly trapped on one of the long-wavelength antenna pigments. Possible spatial organizations of the long-wavelength antenna pigments in the three-dimensional structure of photosystem I are discussed.

Spectroscopic Properties of Reaction Center Pigments in Photosystem II Core Complexes: Revision of the Multimer Model

Absorbance difference spectra associated with the light-induced formation of functional states in photosystem II core complexes from Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 (e.g., P(+)Pheo(-),P(+)Q(A)(-),(3)P) are described quantitatively in the framework of exciton theory. In addition, effects are analyzed of site-directed mutations of D1-His(198), the axial ligand of the special-pair chlorophyll P(D1), and D1-Thr(179), an amino-acid residue nearest to the accessory chlorophyll Chl(D1), on the spectral properties of the reaction center pigments. Using pigment transition energies (site energies) determined previously from independent experiments on D1-D2-cytb559 complexes, good agreement between calculated and experimental spectra is obtained. The only difference in site energies of the reaction center pigments in D1-D2-cytb559 and photosystem II core complexes concerns Chl(D1). Compared to isolated reaction centers, the site energy of Chl(D1) is red-shifted by 4 nm and less inhomogeneously distributed in core complexes. The site energies cause primary electron transfer at cryogenic temperatures to be initiated by an excited state that is strongly localized on Chl(D1) rather than from a delocalized state as assumed in the previously described multimer model. This result is consistent with earlier experimental data on special-pair mutants and with our previous calculations on D1-D2-cytb559 complexes. The calculations show that at 5 K the lowest excited state of the reaction center is lower by approximately 10 nm than the low-energy exciton state of the two special-pair chlorophylls P(D1) and P(D2) which form an excitonic dimer. The experimental temperature dependence of the wild-type difference spectra can only be understood in this model if temperature-dependent site energies are assumed for Chl(D1) and P(D1), reducing the above energy gap from 10 to 6 nm upon increasing the temperature from 5 to 300 K. At physiological temperature, there are considerable contributions from all pigments to the equilibrated excited state P*. The contribution of Chl(D1) is twice that of P(D1) at ambient temperature, making it likely that the primary charge separation will be initiated by Chl(D1) under these conditions. The calculations of absorbance difference spectra provide independent evidence that after primary electron transfer the hole stabilizes at P(D1), and that the physiologically dangerous charge recombination triplets, which may form under light stress, equilibrate between Chl(D1) and P(D1).

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

Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?

Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a approximately 12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield ( approximately 50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by approximately 20-fold and that for closed PS I (with P700 oxidized) is increased by approximately 10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 +/- 1 ps for open PS I and 37 +/- 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing approximately 50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced.

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.