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.     

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.     

Mechanism of electron transfer processes photoinduced by lumazine

UV-A (320-400 nm) and UV-B (280-320 nm) radiation causes damage to DNA and other biomolecules through reactions induced by different endogenous or exogenous photosensitizers. Lumazines are heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. The parent and unsubstituted compound called lumazine (pteridine-2,4(1,3H)-dione; Lum) is able to act as photosensitizer through electron transfer-initiated oxidations. To get further insight into the mechanisms involved, we have studied in detail the oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) photosensitized by Lum in aqueous solution. After UV-A or UV-B excitation of Lum and formation of its triplet excited state ((3)Lum*), three reaction pathways compete for the deactivation of the latter: intersystem crossing to singlet ground state, energy transfer to O(2), and electron transfer between dAMP and (3)Lum* yielding the corresponding pair of radical ions (Lum˙(-) and dAMP˙(+)). In the following step, the electron transfer from Lum˙(-) to O(2) regenerates Lum and forms the superoxide anion (O(2)˙(-)), which undergoes disproportionation into H(2)O(2) and O(2). Finally dAMP˙(+) participates in subsequent reactions to yield products.     

Excited flavin and pterin coenzyme molecules in evolution

Excited flavin and pterin molecules are active in intermolecular energy transfer and in photocatalysis of redox reactions resulting in conservation of free energy. Flavin-containing pigments produced in models of the prebiotic environment are capable of converting photon energy into the energy of phosphoanhydride bonds of ATP. However, during evolution photochemical reactions involving excited FMN or FAD molecules failed to become participants of bioenergy transfer systems, but they appear in enzymes responsible for repair of UV-damaged DNA (DNA photolyases) and also in receptors of blue and UV-A light regulating vital functions of organisms. The families of these photoproteins (DNA-photolyases and cryptochromes, LOV-domain- and BLUF-domain-containing proteins) are different in the structure and in mechanisms of the photoprocesses. The excited flavin molecules are involved in photochemical processes in reaction centers of these photoproteins. In DNA photolyases and cryptochromes the excitation energy on the reaction center flavin is supplied from an antenna molecule that is bound with the same polypeptide. The role of antenna is played by MTHF or by 8-HDF in some DNA photolyases, i.e. also by molecules with known coenzyme functions in biocatalysis. Differences in the structure of chromophore-binding domains suggest an independent origin of the photoprotein families. The analysis of structure and properties of coenzyme molecules reveals some specific features that were significant in evolution for their being selected as chromophores in these proteins.     

Triplet states of bacteriochlorophyll and carotenoids in chromatophores of photosynthetic bacteria

Chromatophores from photosynthetic bacteria were excited with flashes lasting approx. 15 ns. Transient optical absorbance changes not associated with the photochemical electron-transfer reactions were interpreted as reflecting the conversion of bacteriochlorophyll or carotenoids into triplet states. Triplet states of various carotenoids were detected in five strains of bacteria; triplet states of bacteriochlorophyll, in two strains that lack carotenoids. Triplet states of antenna pigments could be distinguished from those of pigments specifically associated with the photochemical reaction centers. Antenna pigments were converted into their triplet states if the photochemical apparatus was oversaturated with light, if the primary photochemical reaction was blocked by prior chemical oxidation of P-870 or reduction of the primary electron acceptor, or if the bacteria were genetically devoid of reaction centers. Only the reduction of the electron acceptor appeared to lead to the formation of triplet states in the reaction centers.In the antenna bacteriochlorophyll, triplet states probably arise from excited singlet states by intersystem crossing. The antenna carotenoid triplets probably are formed by energy transfer from triplet antenna bacteriochlorophyll. The energy transfer process has a half time of approx. 20 ns, and is about 1 × 10³ times more rapid than the reaction of the bacteriochlorophyll triplet states with O₂. This is consistent with a role of carotenoids in preventing the formation of singlet O₂ in vivo. In the absence of carotenoids and O₂, the decay half times of the triplet states are 70 μs for the antenna bacteriochlorophyll and 6–10 μs for the reaction center bacteriochlorophyll. The carotenoid triplets decay with half times of 2–8 μs.With weak flashes, the quantum yields of the antenna triplet states are in the order of 0.02. The quantum yields decline severely after approximately one triplet state is formed per photosynthetic unit, so that even extremely strong flashes convert only a very small fraction of the antenna pigments into triplet states. The yield of fluorescence from the antenna bacteriochlorophyll declines similarly. These observations can be explained by the proposal that singlet-triplet fusion causes rapid quenching of excited singlet states in the antenna bacteriochlorophyll.     

Novel water-soluble photosensitizers from dextrans

Novel water-soluble polymeric photosensitizers based on the natural polymer dextran were synthesized and studied. The modified dextran contained photoactive anthracene (An) chromophores. They were soluble in water with the solubility decreasing with an increase in the number of An moieties bound to the polymeric chain. In aqueous solutions, the macromolecules adopted a compact conformation which resulted in the formation of hydrophobic microdomains. The properties of these domains were characterized with molecular probes such as perylene and pyrazolo-quinoline derivative. The polymer absorbed in the UV/vis region and photosensitized reactions mediated by energy and/or electron transfer from electronically excited An to the molecules of organic compounds solubilized in polymeric microdomains or resided in water.     

Temperature-dependent triplet and fluorescence quantum yields of the photosystem II reaction center described in a thermodynamic model.

A key step in the photosynthetic reactions in photosystem II of green plants is the transfer of an electron from the singlet-excited chlorophyll molecule called P680 to a nearby pheophytin molecule. The free energy difference of this primary charge separation reaction is determined in isolated photosystem II reaction center complexes as a function of temperature by measuring the absolute quantum yield of P680 triplet formation and the time-integrated fluorescence emission yield. The total triplet yield is found to be 0.83 +/- 0.05 at 4 K, and it decreases upon raising the temperature to 0.30 at 200 K. It is suggested that the observed triplet states predominantly arise from P680 but to a minor extent also from antenna chlorophyll present in the photosystem II reaction center. No carotenoid triplet states could be detected, demonstrating that the contamination of the preparation with CP47 complexes is less than 1/100 reaction centers. The fluorescence yield is 0.07 +/- 0.02 at 10 K, and it decreases upon raising the temperature to reach a value of 0.05-0.06 at 60-70 K, increases upon raising the temperature to 0.07 at approximately 165 K and decreases again upon further raising the temperature. The complex dependence of fluorescence quantum yield on temperature is explained by assuming the presence of one or more pigments in the photosystem II reaction center that are energetically degenerate with the primary electron donor P680 and below 60-70 K trap part of the excitation energy, and by temperature-dependent excited state decay above 165 K. A four-compartment model is presented that describes the observed triplet and fluorescence quantum yields at all temperatures and includes pigments that are degenerate with P680, temperature-dependent excited state decay and activated upward energy transfer rates. The eigenvalues of the model are in accordance with the lifetimes observed in fluorescence and absorption difference measurements by several workers. The model suggests that the free energy difference between singlet-excited P680 and the radical pair state P680+l- is temperature independent, and that a distribution of free energy differences represented by at least three values of about 20, 40, and 80 meV, is needed to get an appropriate fit of the data.     

Kinetic studies on the oxidation of cytochrome b 5 Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes

To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.     

The Bakerian lecture, 1984. Biosynthesis of the pigments of life

Many vitally important functions in living systems are carried out by metal ions held as complexes within organic ligands, the organic part of the molecule being a tetrapyrrolic macrocycle. Chlorophyll, haemoglobin, the cytochromes and vitamin B12 all fall into this family of 'pigments of life', a list that emphasizes their central importance in living systems. Research on the biosynthesis of these pigments has involved the synergistic combination of synthesis, structure determination, carbon nuclear magnetic resonance and isotopic labelling with radioactive and stable isotopes in conjunction with enzymology and kinetics. The lecture describes the logical series of experiments based on these approaches which have led to a step-by-step knowledge of the biosynthesis of the parent macrocycle (uroporphyrinogen-III) from which the other pigments are derived. One main pathway from the parent macrocycle involves oxidative transformations and leads eventually to protohaem required inter alia for haemoglobin and myoglobin. The second important pathway makes use of C-methylation to convert the parent macrocycle through many stages finally into vitamin B12. The biosynthetic studies on vitamin B12 are outlined with particular emphasis on the use of isotopic labelling with both radioactive and stable isotopes of carbon and hydrogen. Roughly two-thirds of the entire biosynthetic pathway to vitamin B12 has now been elucidated. The scarcity of several of the known intermediates on the pathway severely hampers future researches and progress towards the total synthesis of these key materials is reviewed. Finally, the lecture brings out the evolutionary interest of what has been discovered about the biosynthesis of the pigments of life.     

The Fragments of the Photosynthetic Electron Transfer Chain in Model Systems

In this paper the recent research from our laboratory is reviewed. Short fragments of the photochemical electron transfer chain of photosynthesis were reproduced in aqueous detergent solutions or in organic solvents. The function of photosystem I is reproduced in a ternary system of chlorophylls, electron donors (dienols, sulfhydryl compounds, hydrazine, etc.), and electron acceptors (viologens, nicotinamide-adenine dinucleotide [NAD], flavines, etc.). Chlorophyll-photosensitized reduction of viologens in some cases is activated by oxygen at the expense of active reductants formed during the photosensitized oxidation of an initial electron donor (thiourea). Chlorophyll-photosensitized oxidoreduction of cytochromes is activated by flavines, viologens, vitamin K derivatives, and some other redox systems (cofactors of cyclic photophosphorylation). The primary mechanism of the reactions studied depends on the reversible chlorophyll photooxidoreduction. In binary systems, chlorophyll (monomeric or aggregated) and electron donor or electron acceptor, reversible photoreduction or photooxidation is observed. Irreversible bacteriochlorophyll oxidation leads to the formation of chlorophyll and protochlorophyll analogues; irreversible protochlorophyll photoreduction results in chlorophyll-like pigment appearance. The photodisaggregation of chlorophyll was observed. The models of photosystem II studied were the photochemical oxygen evolution in aqueous solutions of electron acceptors (ferric compounds, quinone), photosensitized in the near UV part of the spectrum by inorganic semiconductors (tungsten, titanium, and zinc oxides). All reactions described are based on electron (hydrogen) transfer photosensitized by pigment system.     

Kinetics of adrenodoxin reductase oxidation by non-physiologic electron acceptors

The reactions of NADPH oxidation by quinones and inorganic complexes catalyzed by NADPH: adrenodoxin reductase were studied. The catalytic constant for the enzyme at pH 7.0 is 20-25 s-1; the oxidative constants for the quinones vary from 5 X 10(5) to 1.1 X 10(3) M-1 s-1 and show an increase with a rise in the one-electron acceptor reduction potential. The mode of adrenodoxin reductase interaction with oxyquinones differs from that of the enzyme interaction with alkyl-substituted quinones and inorganic complexes. NADPH competitively inhibits electron acceptors, whereas NADP+ is a competitive inhibitor of NADPH and a uncompetitive inhibitor of electron acceptors. (Ki = 25 microM). The depth of FAD incorporation into the enzyme molecule as calculated according to the outer sphere electron transfer theory is 6.1 A.     

Modification of photosystem I reaction center by the extraction and exchange of chlorophylls and quinones.

The photosystem (PS) I photosynthetic reaction center was modified thorough the selective extraction and exchange of chlorophylls and quinones. Extraction of lyophilized photosystem I complex with diethyl ether depleted more than 90% chlorophyll (Chl) molecules bound to the complex, preserving the photochemical electron transfer activity from the primary electron donor P700 to the acceptor chlorophyll A(0). The treatment extracted all the carotenoids and the secondary acceptor phylloquinone (A(1)), and produced a PS I reaction center that contains nine molecules of Chls including P700 and A(0), and three Fe-S clusters (F(X), F(A) and F(B)). The ether-extracted PS I complex showed fast electron transfer from P700 to A(0) as it is, and to FeS clusters if phylloquinone or an appropriate artificial quinone was reconstituted as A(1). The ether-extracted PS I enabled accurate detection of the primary photoreactions with little disturbance from the absorbance changes of the bulk pigments. The quinone reconstitution created the new reactions between the artificial cofactors and the intrinsic components with altered energy gaps. We review the studies done in the ether-extracted PS I complex including chlorophyll forms of the core moiety of PS I, fluorescence of P700, reaction rate between A(0) and reconstituted A(1), and the fast electron transfer from P700 to A(0). Natural exchange of chlorophyll a to 710-740 nm absorbing chlorophyll d in PS I of the newly found cyanobacteria-like organism Acaryochloris marina was also reviewed. Based on the results of exchange studies in different systems, designs of photosynthetic reaction centers are discussed.     

Application of electrochemical kinetics to photosynthesis and oxidative phosphorylation: The redox element hypothesis and the principle of parametric energy coupling

It is suggested that the transfer of electrons within the biological electron transfer chain is subject to the laws of electrochemical kinetics, when membrane-bound electron carriers are involved. Consequently, small tightly bound molecular complexes of two or more electron transfer proteins of different redox potential within an energy transducing membrane, which accept electrons from a donor at one membrane surface and donate it to an acceptor at the other, may be regarded as real and functioning molecular redox elements, which convert the free energy of electrons into electrochemical energy. Especially, the transfer of an electron from excited chlorophyll to an electron acceptor can be looked upon as an electrochemical oxidation of excited chlorophyll at such a complex. In this reaction the electron acceptor complex behaves like a polarized electrode, in which the electrochemical potential gradient is provided by a gradient of redox potential of its constituents.Calculations and qualitative considerations show that this concept leads to a consistent understanding of both primary and secondary reactions in photosynthesis (electron capture, delayed light emission, ion transfer, energy conversion) and can also be applied to oxidative phosphorylation. Within the proposed concept, ion transfer and the development of ion gradients have to be considered as results of electrochemical activity—not as intermediates for energy conversion. For energetic reasons, a non steady state, periodic energy coupling mechanism is postulated which functions by periodic changes of the capacity of the (electrochemically) charged energy transducing membrane, during which capacitive surplus energy is released as chemical energy. Energy transducing membranes may thus be considered as electrochemical parametric energy transformers. This concept explains active periodic conformation changes and mechanochemical processes of energy transducing membranes as energetically essential events, which trigger energy conversion according to the principle of variable parameter energy transformers.The electrochemical approach presented here has been suggested and is supported by the observation, that with respect to electron capture and conversion of excitation energy into electrochemical energy, the behaviour of excited chlorophyll at suitable solid state (semiconductor) electrodes is very similar to that of chlorophyll in photosynthetic reaction centers.     

Photomonomerization of pyrimidine dimers by indoles and proteins.

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wave-lengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases.Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the ¹⁴C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed.Although indoles are able to split the dimers in RNA, they fail to photo-reactivate u.v.-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactivate viral RNA when irradiated at 313 nm, possibly because of side reactions.     

Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile.

1. A system for separation of bile pigments by t.l.c. and for their structure elucidation is presented. Separated bile pigments are characterized by t.l.c. of derived dipyrrolic azopigments. 2. At the tetrapyrrolic stage hydrolysis in strongly alkaline medium followed by t.l.c. demonstrates the presence of bilirubin-IIIalpha, -IXalpha and -XIIIalpha and allows assessment of their relative amounts. 3. Most structural information is derived from analysis of dipyrrolic azopigments. Such derivatives, obtained by treatment of separated bile pigments with diazotized ethyl anthranilate, were separated and purified by t.l.c. Micro methods showed (a) the nature of the dipyrrolic aglycone, (b) the nature of the bonds connecting aglycone to a conjugating group, (c) the ratio of vinyl/isovinyl isomers present in the aglycone and, (d) the nature of the conjugating groups (by suitable derivative formation and t.l.c. with reference to known compounds). 4. In bile of normal dogs at least 20 tetrapyrrolic, diazo-positive bile pigments could be recognized. Except for two pigments the tetrapyrrolic nucleus corresponded predominantly to bilirubin-IXalpha. All conjugated pigments had their conjugating groups connected in ester linkage to the tetrapyrrolic aglycone, Apart from bilirubin-IXalpha, monoconjugates and homogeneous and mixed diconjugates of bilirubin were demonstrated; conjugating groups of major importance were xylose, glucose and glucuronic acid. 5. Bilirubin isomer determination on native bile and isolated bile pigments, and dipyrrole-exchange assays with [14C8]bilirubin indicated (a) that the conjugates pre-exist in bile, and (b) that no significant dipyrrole exchange occurs during isolation of the pigments.     

Evolution of uphill electron transfer

The evolution of photosynthetic energy storage is considered. The primary event in primordial inorganic or organic photoreceptors was charge separation at the expense of light quantum energy. The subsequent improvement of energy storage was attained by separately channeling electrons and “holes” to prevent back reactions. The anisotropic arrangement of photoreceptors in the primary membrane caused a coupling of photochemical charge separation to subsequent ion dislocation and was a prerequisite of primary photophosphorylation. The gradual improvement of the molecular organization of photoreceptor units resulted in antenna and reaction center development. The “hole” was primary located on a peculiar photoreceptor form and the electron passed by tunneling through the chain of intermediate carriers (chlorophylls and pheophytins); thus long-lived charge separation was achieved. The use of the electrons and the “holes” stored in reaction centers for the functioning of the photosynthetic electron transfer chain was realized by cyclic and non-cyclic pathways when the coupling of two photochemical events became the more perfect mechanism to use water molecule as an ultimate electron donor. The appearance of primitive cells inevitably required the coupling of the solar energy conversion mechanism to the reproduction mechanism which used stored solar energy.     

Fluorescence decay kinetics of chlorophyll in photosynthetic membranes

The absorption of light by the pigments of photosynthetic organisms results in electronic excitation that provides the energy to drive the energy-storing light reactions. A small fraction of this excitation gives rise to fluorescence emission, which serves as a sensitive probe of the energetics and kinetics of the excited states. The wavelength dependence of the excitation and emission spectra can be used to characterize the nature of the absorbing and fluorescing molecules and to monitor the process of sensitization of the excitation transfer from one pigment to another. This excitation transfer process can also be followed by the progressive depolarization of the emitted radiation. Using time-resolved fluorescence rise and decay kinetics, measurements of these processes can now be characterized to as short as a few picoseconds. Typically, excitation transfer among the antenna or light harvesting pigments occurs within 100 psec, whereupon the excitation has reached a photosynthetic reaction center capable of initiating electron transport. When this trap is functional and capable of charge separation, the fluorescence intensity is quenched and only rapidly decaying kinetic components resulting from the loss of excitation in transit in the antenna pigment bed are observed. When the reaction centers are blocked or saturated by high light intensities, the photochemical quenching is relieved, the fluorescence intensity rises severalfold, and an additional slower decay component appears and eventually dominates the decay kinetics. This slower (1-2 nsec) decay results from initial charge separation followed by recombination in the blocked reaction centers and repopulation of the excited electronic state, leading to a rapid delayed fluorescence component that is the origin of variable fluorescence. Recent growth in the literature in this area is reviewed here, with an emphasis on new information obtained on excitation transfer, trapping, and communication between different portions of the photosynthetic membranes.     

On the electronic structure of the primary electron donor in bacterial photosynthesis – the bacteriochlorophyll dimer as viewed by EPR/ENDOR methods

The primary electron donor (D) plays a prominent role in electron transfer reactions in the primary processes of photosynthesis. In purple photosynthetic bacteria D is a dimer of bacteriochlorophyll molecules. Investigations on its electronic structure using EPR and ENDOR (electron nuclear double resonance) methods are summarized, focussing on results obtained in the last six years. These encompass studies on the cation radical (D+·) of mutants in which the immediate environment of D is modified through mutagenesis, particularly hydrogen bond and heterodimer mutants. Models using these results to describe the electronic interaction of the dimer halves are discussed. Also, High-Field (95 GHz) EPR to obtain the G-tensor of D+· is addressed. Furthermore, ENDOR on the photoexcited triplet state of D (DT), which in some aspects could serve as a model for the excited singlet state, are discussed. Different approaches towards correlating the electronic structure with function, in particular with the rates of electron transfer reactions, are described.     

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers. Response under conditions of single electron turnover.

Light-induced electric current and potential responses have been measured across planar phospholipid membranes containing reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides. Under conditions in which the reaction centers are restricted to a single electron turnover, the responses can be correlated with the light-induced electron transfer reactions associated with the reaction center. The results indicate that electron transfer from the bacteriochlorophyll dimer to the primary ubiquinone molecule, and from ferrocytochrome c to the oxidized dimer occur in series across the planar membrane. Electron transfer from the primary to secondary ubiquinone molecule is not electrogenic.     

Energy migration in phycobilisomes

Fluorescence emission and polarization spectra of the phycobilisomes (PBS) of the blue-green alga Nostoc muscorum were measured at 20, -73 and -196 degrees C while exciting at the absorption maximum of each pigment in the PBS. The emission spectra were deconvoluted into a number of Gaussian components and energy migration coefficients and quantum yields of fluorescence for the 8 forms of the phycobilins constituting the PBS were calculated. The overlap integrals and the critical and real distances for the energy transfer in the donor-acceptor pairs were evaluated. The general scheme of the energy transfer in the PBS is proposed according to which there is a homogeneous energy migration within each pigment form and a following effective heterogeneous migration directed from the short wavelength forms via the intermediate ones to the terminal long wavelength acceptors. The transfer passes one or more steps of the energy "staircase" which is formed by the excited levels of the forms. The backward "uphill" energy transfer does not take place. These data and the estimates of the real distances of the energy transfer allowed us to make a conclusion on the regular arrangement of the pigments in the PBS, to determine the distances between the chromophores and their localization in a pigment molecule and the distances between the chromophores of different pigments and thus to specify the structure of the PBS.