Kinetics of pigment-acceptor interaction induced by continuous illumination in Synechocystis spaeroides photosystem I preparations cooled to 160 K in the dark and light

The kinetics of dark reduction of chlorophyll P700 oxidized by continuous light in preparations of photosystem I reaction centers from cyanobacterium Synechosystis spharoides cooled in the dark to 160 K is essentially nonexponential. The characteristic times of the components range from fractions of a second to minutes or more. During the cooling of reaction center preparations under illumination with actinic light, most of the chlorophyll P700 molecules are fixed in the oxidized state at 160 K. The kinetics of dark reduction of P700⁺ in the fraction of reaction centers that retain photochemical activity under these conditions is somewhat faster compared to the samples cooled in the dark. A theoretical analysis of substantial deceleration of P700⁺ dark recovery kinetics was done for preparations of photosystem I reaction centers oxidized by continuous light at 160 K in comparison to the experiments where reaction centers were oxidized by short single light flashes. This slowing down of the kinetics in samples excited by continuous illumination can be explained by microconformational relaxation processes related to proton shifts in the reaction center.     

Effect of low temperatures on photochemical activity of PS1 reaction centers from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. frozen under illumination

After cooling of Synechocystis sp. photosystem 1 (PS1) reaction centers (RC) to 160 K under illumination most of the photoactive pigment is fixed for a long time in the oxidized state. The same effect is observed in purple bacteria RC. The dark reduction kinetics of PS1 P700 chlorophyll, which still retains its photochemical activity, in these samples was similar to that in samples cooled in the dark. We suggest that the photoinduced charge separation in PS1 RC, as well as in purple bacteria RC, is accompanied by conformational changes that can be fixed in samples cooled under illumination. As a result, the electrons photomobilized in RC cooled under illumination are unable to return backward the process of electron transfer to P700(+) after cessation of actinic illumination. Such irreversible trapping of electrons can take place in different parts of the PS1 RC electron acceptor chain.     

Electron-transfer kinetics in photosynthetic reaction centers cooled to cryogenic temperatures in the charge-separated state: evidence for light-induced structural changes

We have compared the electron-transfer kinetics in reaction centers (RCs) cooled in the dark with those cooled under illumination (i.e., in the charge-separated state). Large differences between the two cases were observed. We interpreted these findings in terms of light-induced structural changes. The kinetics of charge recombination D+QA-----DQA in RCs containing one quinone were modeled in terms of a distribution of donor-acceptor electron-transfer distances. For RCs cooled under illumination the distribution broadened and shifted to larger distances compared to the distribution for RCs cooled in the dark. The model accounts for the nonexponential decay observed at low temperatures [McElroy, J. D., Mauzerall, D. C., & Feher, G. (1974) Biochim. Biophys. Acta 333, 261-277; Morrison, L.E., & Loach, P.A. (1978) Photochem. Photobiol. 27, 751-757]. A possible physiological role of the structural changes is an enhanced charge stabilization. For RCs with two quinones, the recombination kinetics D+QAQB-----DQAQB were found to be strongly temperature dependent. This was interpreted in terms of temperature-dependent transitions between structural states [Agmon, N., & Hopfield, J.J. (1983) J. Chem. Phys. 78, 6947-6959]. This interpretation requires that these transitions occur at cryogenic temperatures on a time scale t greater than or approximately 10(3) s. The electron transfer from QA- to QB was found to not take place in RCs cooled in the dark (tau ABdark greater than 10(-1) s). In RCs cooled under illumination, we found tau ABlight less than 10(-3) s. We suggest the possibility that the drastic decrease in tau AB observed in RCs cooled under illumination is due to the trapping of a proton near QB-.     

Kinetics of pigment-acceptor interaction induced by steady-state illumination in Synechocystis spaeroides photosystem I preparations cooled to 160 K in the dark and on light

The kinetics of dark reduction of chlorophyll P700 oxidized by steady-state illumination in photosystem I reaction center preparations of cyanobacterium Synechocystis sp. coolled in the dark to 160 K is greatly nonexponential. The characteristic times for the components of the reaction are from fractions of a second to minutes and more. During cooling reaction center preparations on actinic light, a great part of chlorophyll P700 is fixed at 160 K in oxidized state. The kinetics of dark reduction of P700+ in the fraction of reaction centers that retain the photochemical activity in these conditions is faster than the kinetics in samples cooled in the dark. A theoretical analysis of the substantial deceleration of the P700+ dark recovery kinetics was done for photosystem I reaction center preparations oxidized by steady-state illumination to 160 K in contrast with situation that arises after the oxidation of reaction centers by single short light pulses. The deceleration of the kinetics in samples activated by steady-state illumination can be explained by processes of microconformational relaxation, connected with proton shifts in the reaction center structure.     

Effect of energy status of hydrogen bond protons on the rate of electron transfer in photosynthetic reaction centers

We present here a theoretical interpretation of the temperature dependence of the rate of dark recombination which takes place in Rhodobacter sphaeroides reaction centers between a primary quinone (Q(A)) and a bacteriochlorophyll dimer. Taking the energy of interaction between hydrogen bond protons and an excessive electron into account, we described qualitative by this nonmonotonous dependence. We considered a molecular model of the primary quinone from Rb. sphaeroides reaction centers. In addition to the primary quinone, the model includes two reaction center fragments that form hydrogen bonds with Q(A). One of these fragments is His(M219), and the other is the peptide [Asn(M259) - Ala(M260)]. We used the two-center approach with regard for electron-phonon interaction in order to calculate the characteristic time of electron tunneling during the recombination reaction. The energy of the phonon emitted/absorbed during the electron tunneling is determined by a relative shift of the donor and the acceptor energy levels, the detuning of levels. The value of level detuning was shown to be temperature dependent in a nonmonotonous manner in the case of hydrogen bonds with double-well potential energy surface. The characteristic time (or the reaction rate) depends on temperature parametrically. The dependence is nonmonotonous and is in qualitative agreement with the experimental one.     

Energy migration and exciton trapping in green plant photosynthesis

The possible origins of the different fluorescence decay components in green plants are discussed in terms of a random walk and Butler's bipartite model. The interaction of the excitations with the photosystem II reaction centers and, specifically, the regeneration of theses excitations by charge recombination within the reaction centers, are considered. Based on comparisons between fluorescence decay profiles, time-dependent exciton annihilation and photoelectric phenomena, it appears that the fast 200 ps decay component corresponds to primary energy transport from the antenna to the reaction centers and is dominant in filling the photosystem II reaction centers.     

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20°C (nonhardened rye, RNH) or 5°C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.

Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20°C indicated that RNH and RH thylakoids contained the same proportions of fast (α) and slow (β) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for α components increased and the rate constant for β components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5°C.

     

Monomeric bacteriochlorophyll is required for the triplet energy transfer between the primary donor and the carotenoid in photosynthetic bacterial reaction centers

Reaction centers from the carotenoidless mutant Rb. sphaeroides R26 were treated with sodium borohydride which is known to remove one of the accessory monomeric bacteriochlorophylls (BB). Subsequently, the carotenoid, spheroidene, was incorporated into the modified reaction centers. It is demonstrated by optical absorption and circular dichroism experiments that spheroidene, reconstituted into the sodium borohydride-treated Rb. sphaeroides R26 reaction centers, is bound in a single site, in the same environment and with the same structure as spheroidene reconstituted into untreated (native) Rb. sphaeroides R26 reaction centers. Transient optical and electron spin resonance spectroscopic data indicate that unless the accessory BB is present, the primary donor-to-carotenoid triplet energy transfer reaction is inhibited. These observations provide direct evidence for the involvement of the accessory BB in the triplet energy transfer pathway.     

Energy transfers from Photosystem II to Photosystem I in Cryptomonas rufescens (Cryptophyceae)

In Cryptomonas rufescens (Cryptophyceae), phycoerythrin located in the thylakoid lumen is the major accessory pigment. Oxygen action spectra prove phycoerythrin to be efficient in trapping light energy.The fluorescence excitation spectra at ?196°C obtained by the method of Butler and Kitajima (Butler, W.L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72–85) indicate that like in Rhodophycease, chlorophyll a is the exclusive light-harvesting pigment for Photosystem I.For Photosystem II we can observe two types of antennae: (1) a light-harvesting chlorophyll complex connected to Photosystem II reaction centers, which transfers excitation energy to Photosystem I reaction centers when all the Photosystem II traps are closed. (2) A light-harvesting phycoerythrin complex, which transfers excitation energy exclusively to the Photosystem II reaction complexes responsible for fluorescence at 690 nm.We conclude that in Cryptophyceae, phycoerythrin is an efficient light-harvesting pigment, organized as an antenna connected to Photosystem II centers, antenna situated in the lumen of the thylakoid. However, we cannot afford to exclude that a few parts of phycobilin pigments could be connected to inactive chlorophylls fluorescing at 690 nm.     

Means of optimizing light energy conversion in the primary stages of photosynthesis. II. Optimization of the structure of an uniform photosynthetic unit lattice

The possibility of optimization of the structure of a model photosynthetic unit lattice is analysed. The efficiency of the photosynthetic unit operation is evaluated from the time of excitation energy trapping by reaction centers. The calculations assume a F?rster inductive resonance mechanism for energy transfer within light--harvesting antenna and pairwise dipolar interactions. We use the probability matrix method which is adapted to excitation trapping time (but not to excitation jumps number) calculation. It is shown that the specific anisotropy of the distances between antenna molecules (which is in principle possible due to the diskshaped form of chlorophyll molecules) in combination with the optimal spatial arrangement of reaction centers as "well regulated clusters" allows to decrease the time of excitation energy trapping by over an order of magnitude. The requirements for optimization of the structure of a macroscopic photosynthetic unit lattice and the consequences following from them for the in vivo systems are formulated.     

Radical-pair energetics and decay mechanisms in reaction centers containing anthraquinones, naphthoquinones or benzoquinones in place of ubiquinone

In reaction centers from Rhodobacter sphaeroides (formerly called Rhodopseudomonas sphaeroides), light causes an electron-transfer reaction that forms the radical pair state (P+I-, or PF) from the initial excited singlet state (P) of a bacteriochlorophyll dimer (P). Subsequent electron transfer to a quinone (Q) produces the state P+Q-. Back electron transfer can regenerate P from P+Q-, giving rise to 'delayed' fluorescence that decays with approximately the same lifetime as P+Q-. The free-energy difference between P+Q- and P can be determined from the initial amplitude of the delayed fluorescence. In the present work, we extracted the native quinone (ubiquinone) from Rps. sphaeroides reaction centers, and replaced it by various anthraquinones, naphthoquinones, and benzoquinones. We found a rough correlation between the halfwave reduction potential (E1/2) of the quinone used for reconstitution (as measured polarographically in dimethylformamide) and the apparent free energy of the state P+Q- relatively to P. As the E1/2 of the quinone becomes more negative, the standard free-energy gap between P+Q- and P decreases. However, the correlation is quantitatively weak. Apparently, the effective midpoint potentials (Em) of the quinones in situ depend subtly on interactions with the protein environment in the reaction center. Using the value of the Em for ubiquinone determined in native reaction centers as a reference, and the standard free energies determined for P+Q- in reaction centers reconstituted with other quinones, the effective Em values of 12 different quinones in situ are estimated. In native reaction centers, or in reaction centers reconstituted with quinones that give a standard free-energy gap of more than about 0.8 eV between P+Q- and P*, charge recombination from P+Q- to the ground state (PQ) occurs almost exclusively by a temperature-insensitive mechanism, presumably electron tunneling. When reaction centers are reconstituted with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 eV, part or all of the decay proceeds through a thermally accessible intermediate. There is a linear relationship between the log of the rate constant for the decay of P+Q- via the intermediate state and the standard free energy of P+Q-. The higher the free energy, the faster the decay. The kinetic and thermodynamic properties of the intermediate appear not to depend strongly on the quinone used for reconstitution, indicating that the intermediate is probably not simply an activated form of P+Q-.(ABSTRACT TRUNCATED AT 400 WORDS)     

A newly developed SGB buffer greatly enhances energy transfer efficiency from phycobilisomes to photosystem II in cyanobacteria in vitro

The transfer of light energy from phycobilisomes (PBS) to photosystem II (PSII) reaction centers is vital for photosynthesis in cyanobacteria and red algae. To investigate the relationship between PBS and PSII and to optimize the energy transfer efficiency from PBS to PSII, isolation of the PBS-PSII supercomplex is necessary. SPC (sucrose/phosphate/citrate) is a conventional buffer for isolating PBS-PSII supercomplex in cyanobacteria. However, the energy transfer occurring in the supercomplex is poor. Here, we developed a new buffer named SGB by adding 1M glycinebetaine and additional sucrose to SPC buffer. Compared to SPC, the newly developed SGB buffer greatly enhanced the associated populations of PBS with thylakoid membranes and PSII and further improved the energy transfer efficiency from PBS to PSII reaction centers in cyanobacteria in vitro. Therefore, we conclude that SGB is an excellent buffer for isolating the PBS-PSII supercomplex and for enhancing the energy transfer efficiency from PBS to PSII reaction centers in cyanobacteria in vitro.     

Connectivity of Photosystem II Is the Physical Basis of Retrapping in Photosynthetic Thermoluminescence

Energy transfer between photosystem II (PSII) centers is known from previous fluorescence studies. We have studied the theoretical consequences of energetic connectivity of PSII centers on photosynthetic thermoluminescence (TL) and predict that connectivity affects the TL Q band. First, connectivity is expected to make the Q band wider and more symmetric than an ideal first-order TL band. Second, the presence of closed PSII centers in an energetically connected group of PSII centers is expected to lower the probability that an exciton originating in a recombination reaction becomes retrapped. The latter effect would shift the Q band toward lower temperature, and the shift would be greater the higher the percentage of closed PSII centers at the beginning of the measurement. These effects can be generalized as second-order effects, as they make the Q band resemble the second-order TL bands obtained from semiconducting solids. We applied the connected-units model of chlorophyll fluorescence to derive equations for quantifying the second-order effects in TL. To test the effect of the initial proportion of closed reaction centers, we measured the Q band with different intensities of the excitation flash and found that the peak position changed by 2.5°C toward higher temperature when the flash intensity was lowered from saturating to 0.39% of saturating. The result shows that energy transfer between reaction centers of PSII forms the physical basis of retrapping in photosynthetic TL. The second-order effects partially explain the deviation of the form of the Q band from ideal first-order TL.     

The extent of energy transfer among Photosystem II reaction centers in Chlorella

We have investigated the extent of energy transfer among Photosystem II reaction centers in Chlorella vulgaris. The cells show typical non-exponential fluorescence induction kinetics in the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We used single-turnover flashes to determine effective absorption cross-sections for Photosystem II reaction centers (RCII) in cells which were simultaneously illuminated with a continuous background light. We varied the background irradiance to control the fraction, γ, of the total RCII closed at the time of a flash. We found that the absorption cross-section per RCII was almost completely independent of γ. Relative oxygen flash yields measured at low laser flash energies were similarly unaffected when RCII were closed with the background light. We simultaneously measured laseer flash energy saturation curves for oxygen production and for the increase in fluorescence quantum yield measured 30 μs after the laser flash. The oxygen and fluorescence saturation curves were almost identical. We conclude from these results and appropriate theoretical calculations that the difference in the probabilities for escape of excitation energy at open and closed RCII is small (under 0.25). No matter how many RCII share a common antenna, closing RCII does not greatly change the absorption cross-section of the remaining open RCII. Either the probability for escape from closed traps is small, or the probabilities for escape from open and closed traps are nearly equal.     

High-resolution two-dimensional 1H and 14N hyperfine sublevel correlation spectroscopy of the primary quinone of photosystem II

Quinones are naturally occurring isoprenoids that are widely exploited by photosynthetic reaction centers. Protein interactions modify the properties of quinones such that similar quinone species can perform diverse functions in reaction centers. Both type I and type II (oxygenic and nonoxygenic, respectively) reaction centers contain quinone cofactors that serve very different functions as the redox potential of similar quinones can operate at up to 800 mV lower reduction potential when present in type I reaction centers. However, the factors that determine quinone function in energy transduction remain unclear. It is thought that the location of the quinone cofactor, the geometry of its binding site, and the "smart" matrix effects from the surrounding protein environment greatly influence the functional properties of quinones. Photosystem II offers a unique system for the investigation of the factors that influence quinone function in energy transduction. It contains identical plastoquinones in the primary and secondary quinone acceptor sites, Q(A) and Q(B), which exhibit very different functional properties. This study is focused on elucidating the tuning and control of the primary semiquinone state, Q(A)(-), of photosystem II. We utilize high-resolution two-dimensional hyperfine sublevel correlation spectroscopy to directly probe the strength and orientation of the hydrogen bonds of the Q(A)(-) state with the surrounding protein environment of photosystem II. We observe two asymmetric hydrogen bonding interactions of reduced Q(A)(-) in which the strength of each hydrogen bond is affected by the relative nonplanarity of the bond. This study confirms the importance of hydrogen bonds in the redox tuning of the primary semiquinone state of photosystem II.     

High throughput engineering to revitalize a vestigial electron transfer pathway in bacterial photosynthetic reaction centers

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization.     

The involvement of iron and ubiquinone in electron transfer reactions mediated by reaction centers from photosynthetic bacteria.

Reaction centers from Rhodopseudomonas sphaeroides strain R-26 were prepared with varying Fe and ubiquinone (Q) contents. The photooxidation of P-870 to P-870+ was found to occur with the same quantum yield in Fe-depleted reaction centers as in control samples. The kinetics of electron transfer from the initial electron acceptor (I) to Q also were unchanged upon Fe removal. We conclude that Fe has no measurable role in the primary photochemical reaction. The extent of secondary reaction from the first quinone acceptor (QA) to the second quinone acceptor (QB) was monitored by the decay kinetics of P-870+ after excitation of reaction centers with single flashes in the absence of electron donors, and by the amount of P-870 photooxidation that occurred on the second flash in the presence of electron donors. In reaction centers with nearly one iron and between 1 and 2 ubiquinones per reaction center, the amount of secondary electron transfer is proportional to the ubiquinone content above one per reaction center. In reaction centers treated with LiClO4 and o-phenanthroline to remove Fe, the amount of secondary reaction is decreased and is proportional to Fe content. Fe seems to be required for the secondary reaction. In reaction centers depleted of Fe by treatment with SDS and EDTA, the correlation between Fe content and secondary activity is not as good as that found using LiClO4. This is probably due in part to a loss of primary photochemical activity in samples treated with SDS; but the correlation is still not perfect after correction for this effect. The nature of the back reaction between P-870+ and Q-B was investigated using stopped flow techniques. Reaction centers in the P-870+ Q-B state decay with a 1-s half-time in both the presence and absence of o-phenanthroline, an inhibitor of electron transfer between Q-B and QB. This indicates that the back reaction between P-870+ and Q-A is direct, rather than proceeding via thermal repopulation of Q-A. The P-870+ Q-B state is calculated to lie at least 100 mV in free energy below the P-870+ Q-A state.     

Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress

Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.     

On the quenching of the fluorescence yield in photosynthetic systems

A modified matrix model describing transfer of excitation energy in the photosynthetic pigment system is discussed. In addition to the antenna pigments and reaction centers of the simple matrix model, a coupling complex is postulated mediating energy transfer between antenna and reaction centers. The values of the parameters describing the transfer properties of the coupling complex can be chosen in such a way that a number of recent unexplained measurements of fluorescence properties of various purple bacteria can be described. If such coupling complexes are present in oxygen evolving organisms, some of their properties must be different from those of purple bacteria.     

Structural Specificity of Photosynthetic Reaction Centers Provides High Efficiency of Excitation Trapping and Conversion

The atomic structures of photosynthetic reaction centers of two species of purple bacteria and two photosystems 2 of cyanobacteria were resolved in the late last century. In this work I put forward the idea that of the huge body of data available thus far, only three structural factors are responsible for the unique function of conversion of physical energy of electronic excitation into electrochemical energy of separated opposite charges in reaction centers at least in purple bacteria and, perhaps, in other photosynthetic organisms.