Excited chlorophyll and related problems

A historical outline is presented of the primary light energy conversion in photosynthesis studied by our research group. We found that photoexcited chlorophylls, pheophytins and porphyrins are capable of reversible and irreversible oxido-reduction. The mechanism of the photosensitized electron transfer from donor to acceptor molecule is based on the reversible photochemical oxido-reduction of the pigment-sensitizer. This property of the excited pigments is realized in the reaction centres of photosynthetic cells when photooxidation of bacteriochlorophyll(s) or chlorophyll of Photosystem II is coupled to pheophytin reduction leading to the final charge separation.The studies of the state and function of pigments in the course of chlorophyll biosynthesis in cellular and non-cellular systems revealed different monomeric and aggregated forms of pigments and the phenomenon of self-assembly of various forms of chlorophylls, bacteriochlorophylls and protochlorophylls. The discovery of protochlorophyll photoreduction in non-cellular system allowed the study of the molecular mechanisms of this reaction.In order to construct models of photosynthetic charge separation, we used inorganic photocatalysts-semiconductors, mainly titanium dioxide, and pigments incorporated into detergent micelles or lipid vesicles. To prevent back reactions we used heterogeneous systems where primary unstable products were spatially separated; coupling of solubilized chlorophylls or semiconductor particles with bacterial hydrogenase led to molecular hydrogen photoproduction. Light excitation of some coenzymes, mainly NADH and NADPH, was considered from the point of view of early events of chemical evolution.Now we are interested in the creation of photobiochemical systems using principles of photosynthesis for the conversion and storage of solar energy.Written at the invitation of Govindjee.     

Excitation relaxation in the chemically modified photosynthetic reaction center from <Emphasis Type="Italic">Rhodobacter</Emphasis><Emphasis Type="Italic">sphaeroides</Emphasis> 601

The ultrafast excitation relaxation in the sodium borohydride-treated reaction center of Rhodobacter sphaeroides 601 was investigated with selective excitation. From the femtosecond pump-probe measurement at 790 nm, the excitation relaxation demonstrates a biexponential decay with time constants of about 200 fs and 1.4 ps. By comparison with the result from sodium ascorbate-pretreated modified RS601, it could be concluded that the dynamical trace at 790 nm mainly originates from the contribution of accessory bacteriochlorophyll in the active side, and the electrochromic shift arising from the induced positive charge on the special pair primarily affects the absorption band in the red region of the accessory bacteriochlorophyll in RS601. With direct excitation of the special pair, the charge separation and subsequent electron transfer were observed in borohydride-modified RS601. The 2.8 ps component was ascribed to the charge separation and electron transfer from P* to H(A). From the dynamical traces at 790, 800 and 818 nm, the ultrafast energy relaxation from the excited accessory bacteriochlorophyll in the active side is consistent with a two-step energy transfer mechanism. This dynamical observation in modified RS601 is of significance in understanding the physical mechanism of excitation relaxation and energy transfer in the photosynthetic primary process.     

Phase separation,charge separation and biogenesis

The current view of bioenergetics postulates transmembrane charge separation as a primary mechanism of energy storage and transformation. Using that bioenergetic view we examine possible methods of photon driven transduction in primordial vesicles. Two possible types of proton pumps are analyzed and a method of anaerobic photophosphorylation is discussed. Using these principles we theorize about the formation of prebiotic photochemical vesicles utilizing the same transmembrane energy conversions characteristic of contemporary cellular systems.     

Rate constants of primary reversible reactions of electron transfer do not depend on temperature. Numerical experiment

The process of irreversible photochemical charge separation in photosynthetic bacterial reaction centers is proposed to be characterized by the effective rate constant. A formula to compute this effective rate constant is derived. Similar rate constant was previously considered by R.A. Marcus (Marcus R.A. 1956. J. Chem. Phys. 24, 966–978) in order to describe nonphotochemical intermolecular electron transfer. The effective rate constant of the irreversible charge separation in photosynthetic bacterial reaction centers is shown to depend on the temperature. In contrast, rate constants of the forward electron transfer from the excited singlet primary donor to the bacteriochlorophyll and from its ion-radical to the bacteriopheophytin acceptor do not depend on temperature.     

Theoretical study on primary reaction of photosynthetic bacteria

Theoretical calculation was carried out on the primary electron donor P_(870) of photosynthetic bacteria. The results show that: (ⅰ) the bimolecular structure of the primary electron donor is more advantageous in energy than monomolecular structure; (ⅱ) the initial configuration of primary electron donor is no longer stable and changes to the configuration with lower energy and chemical reactivity after the charge separation. In the P_(870), such structural change is completed through the rotation of C_3 acetyl, so the oxygen atom of acetyl interacts with the magnesium atom of another bacterio-chlorophyll molecule, and the total energy and chemical reactivity are reduced evidently. It is suggested that the structural change of the primary electron donor is important in preventing the occurrence of charge recombination during the primary reaction and maintaining the high efficiency of the conversion of sun-light to chemical energy. A new mechanism of primary reaction has been proposed, which can give r     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center     

Chemical evolution of photosynthesis

The principles of biological evolution of photosynthesis are established, but the ways of chemical evolution are unclear yet. The model systems will help to elucidate the problem. Every type of photosynthesis requires photoreceptor absorbing solar radiation. We studied as photoreceptors inorganic components of Earth crust, some coenzymes and porphyrins of abiogenic and biogenic origin. By the aid of inorganic photosensitizers (TiO₂, ZnO) the models of photosystems I and II were constructed.Photochemical activation of some coenzymes may serve as an intermediate step from heterotrophic dark to light metabolism. The further evolutior led to the separation of catalytic and photosensitizing functions. Porphin, chlorin and bacteriochlorin were formed by abiogenic synthesis. Magnesium complexes of porphyrins are active being excited by light. They are capable to reversible acceptance or donation of an electron to partner molecule. Excited Mg-complexes of porphyrins (P) are capable to transfer an electron from electron-donor (D) to electron-acceptor (A) accompanied by conversion of light quanta energy into potential chemical energy.The primary electron transfer unit (D-P-A) was incorporated into primary membrane. The transition from random to anisotropic arrangement of (D-P-A) in the membrane was plausible as a step of evolution: charge translocation appeared. (D-P-A) units created in the period of chemical evolution were probably used in the course of biological evolution. The (D-P-A) units were coupled with noncyclic and cyclic electron transfer resulting in ATP formation; coupling of two (D-P-A) units led to H₂O oxidation and NADP reduction in photosynthetic organisms. The improvement of pigments biosynthesis created the phenomenon of excitation energy migration from the bulk of the pigment to (D-P-A) unit, being creative center. The models described points the plausible steps of chemical evolution; the real sequence of events will be probably disclosed in the studies of precambrian rocks and space exploration.     

Theoretical study on primary reaction of photosynthetic bacteria

Theoretical calculation was carried out on the primary electron donor P₈₇₀ of photosynthetic bacteria. The results show that: (i) the bimolecular structure of the primary electron donor is more advantageous in energy than monomolecular structure; (ii) the initial configuration of primary electron donor is no longer stable and changes to the configuration with lower energy and chemical reactivity after the charge separation. In the P₈₇₀, such structural change is completed through the rotation of C₃ acetyl, so the oxygen atom of acetyl interacts with the magnesium atom of another bacterio-chlorophyll molecule, and the total energy and chemical reactivity are reduced evidently. It is suggested that the structural change of the primary electron donor is important in preventing the occurrence of charge recombination during the primary reaction and maintaining the high efficiency of the conversion of sun-light to chemical energy. A new mechanism of primary reaction has been proposed, which can give reasonable explanations to the results of kinetic and site mutation studies.     

Towards artificial photosynthesis: ruthenium-manganese chemistry mimicking photosystem II reactions

The reaction center Photosystem II is a key component of the most successful solar energy converting machinery on earth: the oxygenic photosynthesis. Photosystem II uses light to drive the reduction of plastoquinone and the oxidation of water. Water-oxidation is catalyzed by a manganese cluster and gives the organism an abundant source of electrons. The principles of photosynthesis have inspired chemists to mimic these reactions in artificial molecular assemblies. Synthetic light-harvesting antennae and light-induced charge separation systems have been demonstrated by several groups. More recently, there has been an increasing effort to mimic Photosystem II by coupling light-driven charge separation to water oxidation, catalyzed by synthetic manganese complexes.     

Structure, function and regulation of plant photosystem I

Photosystem I (PSI) is a multisubunit protein complex located in the thylakoid membranes of green plants and algae, where it initiates one of the first steps of solar energy conversion by light-driven electron transport. In this review, we discuss recent progress on several topics related to the functioning of the PSI complex, like the protein composition of the complex in the plant Arabidopsis thaliana, the function of these subunits and the mechanism by which nuclear-encoded subunits can be inserted into or transported through the thylakoid membrane. Furthermore, the structure of the native PSI complex in several oxygenic photosynthetic organisms and the role of the chlorophylls and carotenoids in the antenna complexes in light harvesting and photoprotection are reviewed. The special role of the 'red' chlorophylls (chlorophyll molecules that absorb at longer wavelength than the primary electron donor P700) is assessed. The physiology and mechanism of the association of the major light-harvesting complex of photosystem II (LHCII) with PSI during short term adaptation to changes in light quality and quantity is discussed in functional and structural terms. The mechanism of excitation energy transfer between the chlorophylls and the mechanism of primary charge separation is outlined and discussed. Finally, a number of regulatory processes like acclimatory responses and retrograde signalling is reviewed with respect to function of the thylakoid membrane. We finish this review by shortly discussing the perspectives for future research on PSI.     

Virtual intermediates in photosynthetic electron transfer.

We explore the possibility of virtual transfer in the primary charge separation of photosynthetic bacteria within the context of several types of experimental data. We show that the peak that might be expected in the virtual rate as electric fields vary the intermediate state energy is severely broadened by coupling to high-frequency modes. The Stark absorption kinetics data are thus consistent with virtual transfer in the primary charge separation. High-frequency coupling also makes the temperature dependence weak over a wide range of parameters. We demonstrate that Stark fluorescence anisotropy data, usually taken as evidence of virtual transfer, can in fact be consistent with two-step transfer. We suggest a two-pulse excitation experiment to quantify the contributions from two-step and virtual transfer. We show that virtual absorption into a charge transfer state can make a substantial contribution to the Stark absorption spectrum in a way that is not related to any derivative of the absorption spectrum.     

Structure, function and regulation of plant photosystem I

Photosystem I (PSI) is a multisubunit protein complex located in the thylakoid membranes of green plants and algae, where it initiates one of the first steps of solar energy conversion by light-driven electron transport. In this review, we discuss recent progress on several topics related to the functioning of the PSI complex, like the protein composition of the complex in the plant Arabidopsis thaliana, the function of these subunits and the mechanism by which nuclear-encoded subunits can be inserted into or transported through the thylakoid membrane. Furthermore, the structure of the native PSI complex in several oxygenic photosynthetic organisms and the role of the chlorophylls and carotenoids in the antenna complexes in light harvesting and photoprotection are reviewed. The special role of the ‘red’ chlorophylls (chlorophyll molecules that absorb at longer wavelength than the primary electron donor P700) is assessed. The physiology and mechanism of the association of the major light-harvesting complex of photosystem II (LHCII) with PSI during short term adaptation to changes in light quality and quantity is discussed in functional and structural terms. The mechanism of excitation energy transfer between the chlorophylls and the mechanism of primary charge separation is outlined and discussed. Finally, a number of regulatory processes like acclimatory responses and retrograde signalling is reviewed with respect to function of the thylakoid membrane. We finish this review by shortly discussing the perspectives for future research on PSI.     

Fundamental limits on wavelength, efficiency and yield of the charge separation triad

In an attempt to optimize a high yield, high efficiency artificial photosynthetic protein we have discovered unique energy and spatial architecture limits which apply to all light-activated photosynthetic systems. We have generated an analytical solution for the time behavior of the core three cofactor charge separation element in photosynthesis, the photosynthetic cofactor triad, and explored the functional consequences of its makeup including its architecture, the reduction potentials of its components, and the absorption energy of the light absorbing primary-donor cofactor. Our primary findings are two: First, that a high efficiency, high yield triad will have an absorption frequency more than twice the reorganization energy of the first electron transfer, and second, that the relative distance of the acceptor and the donor from the primary-donor plays an important role in determining the yields, with the highest efficiency, highest yield architecture having the light absorbing cofactor closest to the acceptor. Surprisingly, despite the increased complexity found in natural solar energy conversion proteins, we find that the construction of this central triad in natural systems matches these predictions. Our analysis thus not only suggests explanations for some aspects of the makeup of natural photosynthetic systems, it also provides specific design criteria necessary to create high efficiency, high yield artificial protein-based triads.     

Reorganization energy of the initial electron-transfer step in photosynthetic bacterial reaction centers.

The reorganization energy (lambda) for electron transfer from the primary electron donor (P*) to the adjacent bacteriochlorophyll (B) in photosynthetic bacterial reaction centers is explored by molecular-dynamics simulations. Relatively long (40 ps) molecular-dynamics trajectories are used, rather than free energy perturbation techniques. When the surroundings of the reaction center are modeled as a membrane, lambda for P* B --> P+ B- is found to be approximately 1.6 kcal/mol. The results are not sensitive to the treatment of the protein's ionizable groups, but surrounding the reaction center with water gives higher values of lambda (approximately 6.5 kcal/mol). In light of the evidence that P+ B- lies slightly below P* in energy, the small lambda obtained with the membrane model is consistent with the speed and temperature independence of photochemical charge separation. The calculated reorganization energy is smaller than would be expected if the molecular dynamics trajectories had sampled the full conformational space of the system. Because the system does not relax completely on the time scale of electron transfer, the lambda obtained here probably is more pertinent than the larger value that would be obtained for a fully equilibrated system.     

Some experiments on the primary electron acceptor in reaction centres from Rhodopseudomonas sphaeroides

The bacterial reaction center absorbance change at 450 nm (A-450) assigned to an anionic semiquinone, has been suggested as a candidate for the reduced form of the primary electron acceptor in bacterial photosynthesis. In reaction centers of Rhodopseudomonas sphaeroides we have found kinetic discrepancies between the decay of A-450 and the recovery of photochemical competence. In addition, no proton uptake is measurable on the first turnover, although subsequent ones elicit one proton bound per electron. These results are taken to indicate that the acceptor reaction after a long dark period may be different for the first turnover than for subsequent ones. It is suggested that A-450 is still a likely candidate for the acceptor function but that in reaction centers, additional quinone may act as an adventitious primary acceptor when the “true” primary acceptor is reduced. Alternatively, the primary acceptor may act in a “ping-pong” fashion with respect to subsequent photoelectrons.     

The role of electrostatics in proton-conducting membrane protein complexes

Electrostatic interactions play a key role in the coupling of electron and proton transfer in membrane protein complexes during the conversion of the energy stored in sunlight or reduced substrates into biochemical energy via a transmembrane electrochemical proton potential. Principles of charge stabilization within membrane proteins are reviewed and discussed for photosynthetic reaction centers, cytochrome c oxidases, and diheme-containing quinol:fumarate reductases. The impact of X-ray structure-based electrostatic calculations on the functional interpretation of these structural coordinates, on providing new explanations for experimental observations, and for the design of more focused additional experiments is illustrated by a number of key examples.     

Usefulness of thermoluminescence in herbicide research

Many herbicides of different chemical structure inhibit photosynthetic electron flow by interrupting the photosyn‐thetic electron flow by interrupting the photosynthetic electron transport chain between the primary acceptor (Q_A) and the secondary acceptor (Q_B) of photosystem 2 (PS2). Thermoluminescence (TL) originates from PS2, and the bands of the glow curve can be related to the charge recombination between positively charged donors and negatively charged acceptors. The glow curve of TL is strongly influenced by addition of PS2 herbicides. The herbicide treatment shifts the peak position and activation energy of the TL band related to Q_A, suggesting that herbicide binding affects the midpoint redox potential not only of Q _B but also that of Q_A. On the basis of the band shift the herbicides of various chemical structures can be classified into different “thermodynamical” groups which relfect the differences in the binding properties of these herbicides. As a new approach TL seems to be a useful technique in studying the mechanism and site of action of herbicides that inhibit electron transport of PS2.     

The first catalytic step of the light-driven enzyme protochlorophyllide oxidoreductase proceeds via a charge transfer complex

In chlorophyll biosynthesis protochlorophyllide reductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide) to chlorophyllide, providing a rare opportunity to trap and characterize catalytic intermediates at low temperatures. Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR. Different models involving the formation of either radical species or charge transfer complexes have been proposed for the initial photochemical step, which forms a nonfluorescent intermediate absorbing at 696 nm (A696). Our EPR data show that the concentration of the radical species formed in the initial photochemical step is not stoichiometric with conversion of substrate. Instead, a large Stark effect, indicative of charge transfer character, is associated with A696. Two components were required to fit the Stark data, providing clear evidence that charge transfer complexes are formed during the initial photochemistry. The temperature dependences of both A696 formation and NADPH oxidation are identical, and we propose that formation of the A696 state involves hydride transfer from NADPH to form a charge transfer complex. A catalytic mechanism of POR is suggested in which Pchlide absorbs a photon, creating a transient charge separation across the C-17-C-18 double bond, which promotes ultrafast hydride transfer from the pro-S face of NADPH to the C-17 of Pchlide. The resulting A696 charge transfer intermediate facilitates transfer of a proton to the C-18 of Pchlide during the subsequent first "dark" reaction.     

Light Harvesting and State Transitions in Cyanobacteria

Cyanobacteria are oxygenic phototrophic prokaryotes and are considered to be the ancestors of chloroplasts. Their photosynthetic machinery is functionally equivalent in terms of primary photochemistry and photosynthetic electron transport. Fluorescence measurements and other techniques indicate that cyanobacteria, like plants, are capable of redirecting pathways of excitation energy transfer from light harvesting antennae to both photosystems. Cyanobacterial cells can reach two energetically different states, which are defined as “State 1” (obtained after preferential excitation of photosystem I) and “State 2” (preferential excitation of photosystem II). These states can be distinguished by static and time resolved fluorescence techniques. One of the most important conclusions reached so far is that the presence of both photosystems, as well as certain antenna components, are necessary for state transitions to occur. Spectroscopic evidence suggests that changes in the coupling state of the light harvesting antenna complexes (the phycobilisomes) to both photosystems occur during state transitions. The finding that the phycobilisome complexes are highly mobile on the surface of the thylakoid membrane (the mode of interaction with the thylakoid membrane is essentially unknown), has led to the proposal that they are in dynamic equilibrium with both photosystems and regulation of energy transfer is mediated by changes in affinity for either photosystem.     

Parameters affecting the chemical work output of a hybrid photoelectrochemical biofuel cell.

A hybrid photoelectrochemical biofuel cell employing the photoanode architecture of a dye-sensitized solar cell has been assembled. A porphyrin dye sensitizes a TiO(2) semiconductor over the visible range to beyond 650 nm. Photoinduced charge separation at the dye-TiO(2) interface results in electron migration to a cathode, and the holes generated on surface bound dyes oxidize soluble electron mediators. The increased [Ox] : [Red] ratio of the mediator drives the solution-based enzymatic oxidation of appropriate substrates. In this report we investigate how the accumulation of anodic and cathodic products limits cell performance. The NAD(+)/NADH and benzoquinone/hydroquinone redox couples were studied as sacrificial electron donors in the absence of appropriate enzymes or substrates. Comparatively poor cell performance was observed using the benzoquinone/hydroquinone couple. This effect is explained in terms of rapid charge recombination by electron donation from the electrode to benzoquinone in solution, as compared to much less recombination with NAD(+). With the NAD(+)/NADH couple the cell performance is relatively independent of the redox poise of the anode solution, but limited by accumulation of reduction products in the cathodic compartment. Using the NAD(+)/NADH couple, the photochemical reforming of ethanol to hydrogen was demonstrated under conditions where the process would be endergonic in the dark.