Excitation energy transfer and trapping dynamics in the core complex of the filamentous photosynthetic bacterium Roseiflexus castenholzii

The light-harvesting core complex of the thermophilic filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii is intrinsic to the cytoplasmic membrane and intimately bound to the reaction center (RC). Using ultrafast transient absorption and time-resolved fluorescence spectroscopy with selective excitation, energy transfer, and trapping dynamics in the core complex have been investigated at room temperature in both open and closed RCs. Results presented in this report revealed that the excited energy transfer from the BChl 800 to the BChl 880 band of the antenna takes about 2?ps independent of the trapping by the RC. The time constants for excitation quenching in the core antenna BChl 880 by open and closed RCs were found to be 60 and 210?ps, respectively. Assuming that the light harvesting complex is generally similar to LH1 of purple bacteria, the possible structural and functional aspects of this unique antenna complex are discussed. The results show that the core complex of Roseiflexus castenholzii contains characteristics of both purple bacteria and Chloroflexus aurantiacus.     

Ultrafast transient absorption studies on Photosystem I reaction centers from Chlamydomonas reinhardtii. 1. A new interpretation of the energy trapping and early electron transfer steps in Photosystem I

The energy transfer and charge separation kinetics in core Photosystem I (PSI) particles of Chlamydomonas reinhardtii has been studied using ultrafast transient absorption in the femtosecond-to-nanosecond time range. Although the energy transfer processes in the antenna are found to be generally in good agreement with previous interpretations, we present evidence that the interpretation of the energy trapping and electron transfer processes in terms of both kinetics and mechanisms has to be revised substantially as compared to current interpretations in the literature. We resolved for the first time i), the transient difference spectrum for the excited reaction center state, and ii), the formation and decay of the primary radical pair and its intermediate spectrum directly from measurements on open PSI reaction centers. It is shown that the dominant energy trapping lifetime due to charge separation is only 6-9 ps, i.e., by a factor of 3 shorter than assumed so far. The spectrum of the first radical pair shows the expected strong bleaching band at 680 nm which decays again in the next electron transfer step. We show furthermore that the early electron transfer processes up to approximately 100 ps are more complex than assumed so far. Several possibilities are discussed for the intermediate redox states and their sequence which involve oxidation of P700 in the first electron transfer step, as assumed so far, or only in the second electron transfer step, which would represent a fundamental change from the presently assumed mechanism. To explain the data we favor the inclusion of an additional redox state in the electron transfer scheme. Thus we distinguish three different redox intermediates on the timescale up to 100 ps. At this level no final conclusion as to the exact mechanism and the nature of the intermediates can be drawn, however. From comparison of our data with fluorescence kinetics in the literature we also propose a reversible first charge separation step which has been excluded so far for open PSI reaction centers. For the first time an ultrafast 150-fs equilibration process, occurring among exciton states in the reaction center proper, upon direct excitation of the reaction center at 700 nm, has been resolved. Taken together the data call for a fundamental revision of the present understanding of the energy trapping and early electron transfer kinetics in the PSI reaction center. Due to the fact that it shows the fastest trapping time observed so far of any intact PSI particle, the PSI core of C. reinhardtii seems to be best suited to further characterize the electron transfer steps and mechanisms in the reaction center of PSI.     

Efficient energy transfer from the carotenoid S(2) state in a photosynthetic light-harvesting complex

Previously, the spatial arrangement of the carotenoid and bacteriochlorophyll molecules in the peripheral light-harvesting (LH2) complex from Rhodopseudomonas acidophila strain 10050 has been determined at high resolution. Here, we have time resolved the energy transfer steps that occur between the carotenoid's initial excited state and the lowest energy group of bacteriochlorophyll molecules in LH2. These kinetic data, together with the existing structural information, lay the foundation for understanding the detailed mechanisms of energy transfer involved in this fundamental, early reaction in photosynthesis. Remarkably, energy transfer from the rhodopin glucoside S(2) state, which has an intrinsic lifetime of approximately 120 fs, is by far the dominant pathway, with only a minor contribution from the longer-lived S(1) state.     

Bridging the gap between structural and lattice models: a parameterization of energy transfer and trapping in Photosystem I

In the absence of an accurate structural model, the excited state dynamics of energy-transferring systems are often modeled using lattice models. To demonstrate the validity and other potential merits of such an approach we present the results of the modeling of the energy transfer and trapping in Photosystem I based upon the 2.5 A structural model, and show that these results can be reproduced in terms of a lattice model with only a few parameters. It has recently been shown that at room temperature the dynamics of a hypothetical Photosystem I particle, not containing any red chlorophylls (chls), are characterized by a longest (trapping) lifetime of 18 ps. The structure-based modeling of the dynamics of this particle yields an almost linear relationship between the possible values of the intrinsic charge-separation time at P700, 1/gamma, and the average single-site lifetime in the antenna, tauss. Lattice-based modeling, using the approach of a perturbed two-level model, reproduces this linear relation between tauss and 1/gamma. Moreover, this approach results in a value of the (modified) structure-function corresponding to a structure exhibiting a mixture of the characteristics of both a square and a cubic lattice, consistent with the structural model. These findings demonstrate that the lattice model describes the dynamics of the system appropriately. In the lattice model, the total trapping time is the sum of the delivery time to the reaction center and the time needed to quench the excitation after delivery. For the literature value of tauss=150 fs, both these times contribute almost equally to the total trapping time of 18 ps, indicating that the system is neither transfer- nor trap-limited. The value of approximately 9 ps for the delivery time is basically equal to the excitation-transfer time from the bulk chls to the red chls in Synechococcus elongatus, indicating that energy transfer from the bulk to the reaction center and to the red chls are competing processes. These results are consistent with low-temperature time-resolved and steady-state fluorescence measurements. We conclude that lattice models can be used to describe the global energy-transfer properties in complex chromophore networks, with the advantage that such models deal with only a few global, intuitive parameters rather than the many microscopic parameters obtained in structure-based modeling.     

Spectral inhomogeneity of photosystem I and its influence on excitation equilibration and trapping in the cyanobacterium Synechocystis sp. PCC6803 at 77 K.

Ultrafast transient absorption spectroscopy was used to probe excitation energy transfer and trapping at 77 K in the photosystem I (PSI) core antenna from the cyanobacterium Synechocystis sp. PCC 6803. Excitation of the bulk antenna at 670 and 680 nm induces a subpicosecond energy transfer process that populates the Chl a spectral form at 685--687 nm within few transfer steps (300--400 fs). On a picosecond time scale equilibration with the longest-wavelength absorbing pigments occurs within 4-6 ps, slightly slower than at room temperature. At low temperatures in the absence of uphill energy transfer the energy equilibration processes involve low-energy shifted chlorophyll spectral forms of the bulk antenna participating in a 30--50-ps process of photochemical trapping of the excitation by P(700). These spectral forms might originate from clustered pigments in the core antenna and coupled chlorophylls of the reaction center. Part of the excitation is trapped on a pool of the longest-wavelength absorbing pigments serving as deep traps at 77 K. Transient hole burning of the ground-state absorption of the PSI with excitation at 710 and 720 nm indicates heterogeneity of the red pigment absorption band with two broad homogeneous transitions at 708 nm and 714 nm (full-width at half-maximum (fwhm) approximately 200--300 cm(-1)). The origin of these two bands is attributed to the presence of two chlorophyll dimers, while the appearance of the early time bleaching bands at 683 nm and 678 nm under excitation into the red side of the absorption spectrum (>690 nm) can be explained by borrowing of the dipole strength by the ground-state absorption of the chlorophyll a monomers from the excited-state absorption of the dimeric red pigments.     

Effects of spectral variety and molecular orientation on energy trapping in the photosynthetic unit: a model calculation

It is proposed that different spectral varieties of chlorophylls exist in the photosynthetic unit of green plants in order to accelerate the transfer of excitation energy to the reaction center, and thus allow the operation of physically larger units with greater light-harvesting power. This proposal is supported by computer calculation of trapping probabilities on model arrays containing three spectral forms of antenna chlorophyll in addition to reaction center chlorophyll. The calculations assume nearest neighbor transfer steps only, and pairwise dipolar interactions of the sort that feature in the inductive resonance mechanism of Förster. Mutual orientation of transition moment vectors also affects the number of jumps and the time needed for energy trapping. Spectral variety increases the trapping rate by a factor of 4 to 5 over the uniform chlorophyll rate on the arrays examined, and the proper use of orientation may introduce a similar factor. The arguments here provide a plausible explanation not only for the existence of the more abundant forms of chlorophyll a, but also for the specialized form “C705” and for chlorophyll b.     

The photochemical trapping rate from red spectral states in PSI-LHCI is determined by thermal activation of energy transfer to bulk chlorophylls

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)-light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the "bulk" acceptor chlorophyll (chl) states in the framework of the Arrhenius-Eyring theory. It is shown that the wavelength-dependent "activation energies" come out in the range between 1.35 and 2.7 kcal mol(-1), increasing with the emission wavelength within the interval 710-770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130-280 K for PSI-LHCI. We conclude that the variable trapping time in PSI-LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.     

Time-resolved absorption and emission show that the CP43' antenna ring of iron-stressed synechocystis sp. PCC6803 is efficiently coupled to the photosystem I reaction center core

Excitation energy transfer and trapping processes in an iron stress-induced supercomplex of photosystem I from the cyanobacterium Synechocystis sp. PCC6803 were studied by time-resolved absorption and fluorescence spectroscopy on femtosecond and picosecond time scales. The data provide evidence that the energy transfer dynamics of the CP43'-PSI supercomplex are consistent with energy transfer processes that occur in the Chl a network of the PSI trimer antenna. The most significant absorbance changes in the CP43'-PSI supercomplex are observed within the first several picoseconds after the excitation into the spectral region of CP43' absorption (665 nm). The difference time-resolved spectra (DeltaDeltaA) resulting from subtraction of the PSI trimer kinetic data from the CP43'-PSI supercomplex data indicate three energy transfer processes with time constants of 0.2, 1.7, and 10 ps. The 0.2 ps kinetic phase is tentatively interpreted as arising from energy transfer processes originating within or between the CP43' complexes. The 1.7 ps phase is interpreted as possibly arising from energy transfer from the CP43' ring to the PSI trimer via closely located clusters of Chl a in CP43' and the PSI core, while the slower 10 ps process might reflect the overall excitation transfer from the CP43' ring to the PSI trimer. These three fast kinetic phases are followed by a 40 ps overall excitation decay in the supercomplex, in contrast to a 25 ps overall decay observed in the trimer complex without CP43'. Excitation of Chl a in both the CP43'-PSI antenna supercomplex and the PSI trimer completely decays within 100 ps, resulting in the formation of P700(+). The data indicate that there is a rapid and efficient energy transfer between the outer antenna ring and the PSI reaction center complex.     

The assembly of protein subunits and cofactors in photosystem I

The recently determined crystal structures of photosystems I and II at 2.5 A and 3.8 A resolution, respectively, have improved the structural basis for understanding the processes of light trapping, exciton transfer and electron transfer occurring in the primary steps of oxygenic photosynthesis. Understanding the assembly of the 12 protein subunits and 128 cofactors in photosystem I allows us to study the possible functions of the individual players in this protein-cofactor complex.     

The photochemical trapping rate from red spectral states in PSI–LHCI is determined by thermal activation of energy transfer to bulk chlorophylls

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)–light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the “bulk” acceptor chlorophyll (chl) states in the framework of the Arrhenius–Eyring theory. It is shown that the wavelength-dependent “activation energies” come out in the range between 1.35 and 2.7 kcal mol⁻¹, increasing with the emission wavelength within the interval 710–770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130–280 K for PSI–LHCI. We conclude that the variable trapping time in PSI–LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.     

Rat polymerase beta binds double-stranded DNA using exclusively the 8-kDa domain. Stoichiometries,intrinsic affinities,and cooperativities

Analyses of the interactions of rat polymerase beta (rat pol beta) with a double-stranded DNA have been performed using the quantitative fluorescence titration and fluorescence energy transfer techniques. The obtained results show that rat pol beta binds to dsDNA oligomers with the site-size of the enzyme-dsDNA complex n = 5 +/- 1 base pairs. The small site-size of the complex is a consequence of engagement of only the 8-kDa domain in intrinsic interactions with the dsDNA. This conclusion is directly supported by the fluorescence energy transfer between the single tryptophan residue on the 31-kDa domain and fluorescence acceptor located on the DNA. The dsDNA oligomer is bound at a distance of at least 55 A from the tryptophan, excluding the 31-kDa domain from any closed contact with the DNA. Moreover, in the complex with the dsDNA, the enzyme is bound in "open" conformational state. The intrinsic interactions are accompanied by a net release of about four to five ions. The net ion release is dominated by cations as a result of the exclusive engagement of the 8-kDa domain in interactions. Magnesium affects the net ion release through direct binding of Mg(2+) cations to the protein. Surprisingly, binding of rat pol beta to the dsDNA is characterized by strong positive cooperative interactions, a very different behavior from that previously observed for pol beta complexes with the ssDNA and gapped DNAs. Contrary to intrinsic affinities, cooperative interactions are accompanied by a net uptake of about three to five ions. Anions have a large contribution to the net ion uptake, indicating that cooperative interactions characterize protein-protein interactions. The significance of these results for the pol beta functioning in damaged-DNA recognition processes is discussed.     

Relationship between chlorophyll concentration and the energy reaction between protochlorophyll and chlorophyll in mixed associations

In the paper results are presented of investigation of protochlorophyll (PChl) and chlorophyll (Chl) mixed associations and of interaction between them within the polymer molecular complex, which forms in mixture of water-dioxane (1 : 4). The initial PChl concentration in all solutions was constant (CPChl = 1 . 10(-5) m/l), and Chl concentration varied from 1.10(-8) m/l up to 2.10(-5) m/l. It is shown that with the rise of Chl proportion in the mixed aggregate the rearrangement of both donor (PChl) and acceptor (Chl) components of complex takes place. The luminescence quenching of PChl and the sensitization of Chl emission in mixed pigment associates were investigated of different Chl levels and the evaluation of efficiency of intracomplex electronic excitation energy transfer, determined from quenching and sensibilization, was performed. Similar dependence of energy transfer effectiveness on Chl concentration, determined by the two above-mentioned methods shows that the excitation migration in an associate takes place without losses. An analysis of results permits to conclude that a small trapping efficiency of PChl excitation by the acceptor part of the complex may be connected with the existence of the prerelaxation reverse energy transfer from Chl to PChl in mixed pigment associates. On the basis of the obtained data a mechanism of energy transfer from protochlorophyllide to chlorophyllide in etiolated leaves and homogenates is discussed.     

Reaction profile of the last step in cytochrome P-450 catalysis revealed by studies of model complexes

 A series of oxoiron(IV) porphyrin cation radical complexes was investigated as compound I analogs of cytochrome P-450. Both the spectroscopic features and the reactivities of the complexes in oxygen atom transfer to olefins were examined as a function of only one variable, the axial ligand trans to the oxoiron(IV) bond. The results disclosed two important kinetic steps – electron transfer from olefin to oxoiron(IV) and intramolecular electron transfer from metal to porphyrin radical – which are affected differently by the axial ligands. The large kinetic barrier of the latter step in the reaction of olefins with the perchlorato-bound oxoiron(IV) porphyrin cation radical complex enabled the trapping of a reaction intermediate in which the metal, but not the porphyrin radical, is reduced. The first electron transfer step is probably followed by σ-bond formation, which readily accounts for formation of isomerized organic products at low temperatures. It is finally postulated that part of the enhanced oxygenation activities of cytochrome P-450 monooxygenases and chloroperoxidases is due to a lowering of the energy barrier for the second electron transfer step via participation of their redox-active cysteinate ligand. Received: 16 January 1997 / Accepted: 24 May 1997     

Kinetic analysis of R67 dihydrofolate reductase folding: from the unfolded monomer to the native tetramer

R67 dihydrofolate reductase (DHFR) is a homotetrameric enzyme. Its subunit has a core structure consisting of five antiparallel beta-strands that form a compact beta-barrel. Our interest was to describe the molecular mechanism of the complete folding pathway of this beta-sheet protein, focusing on how the oligomerization steps are coordinated with the formation of secondary and tertiary structures all along the folding process. The folding kinetics of R67 dihydrofolate reductase into dimers at pH 5.0 were first examined by intrinsic tryptophan fluorescence, fluorescence energy transfer, and circular dichroism spectroscopy. The process was shown to consist of at least four steps, including a burst, a rapid, a medium, and a slow phase. Measurements of the ellipticity at 222 nm indicated that about 50% of the total change associated with refolding occurred during the 4 ms dead time of the stopped-flow instrument, indicating a substantial burst of secondary structure. The bimolecular association step was detected using fluorescence energy transfer and corresponded to the rapid phase. The slow phase was attributed to a rate-limiting isomerization of peptidyl-prolyl bonds involving 15% of the unfolded population. A complete folding pathway from the unfolded monomer to the native tetramer was proposed and an original model based upon the existence of early partially folded monomeric intermediates, rapidly stabilized in a dimeric form able to self-associate into the native homotetramer was formulated. The rate constants of these various steps were determined by fitting the kinetic traces to this model and supported our mechanistic assumptions.     

A general model for regulation of photosynthetic unit function by protein phosphorylation

We propose that regulatory effects of membrane protein phosphorylation in photosynthetic systems result in all cases from simultaneous phosphorylation by a single kinase of the polypeptides of two intrinsic pigment-protein complexes, with phosphorylation leading to their mutual electrostatic repulsion in a direction parallel to the membrane plane and therefore to decreased excitation energy transfer between them. One complex is a peripheral light-harvesting complex and the other is bound to the reaction centre and functions as a link in excitation energy transfer. Immediate effects of phosphorylation are therefore decreased absorption cross-section together with decreased cooperativity of photosynthetic units. This general model applies equally to photosystem II of green plants, algae and cyanobacteria, as well as to the single photosystem of purple bacteria. Special cases of this general model permit increased excitation energy transfer to one type of reaction centre at the expense of another, and this may occur even in laterally homogeneous membranes that are uniformly unappressed.     

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.     

Photosystem II: The machinery of photosynthetic water splitting

This review summarizes our current state of knowledge on the structural organization and functional pattern of photosynthetic water splitting in the multimeric Photosystem II (PS II) complex, which acts as a light-driven water: plastoquinone-oxidoreductase. The overall process comprises three types of reaction sequences: (1) photon absorption and excited singlet state trapping by charge separation leading to the ion radical pair [Formula: see text] formation, (2) oxidative water splitting into four protons and molecular dioxygen at the water oxidizing complex (WOC) with P680+* as driving force and tyrosine Y(Z) as intermediary redox carrier, and (3) reduction of plastoquinone to plastoquinol at the special Q(B) binding site with Q(A)-* acting as reductant. Based on recent progress in structure analysis and using new theoretical approaches the mechanism of reaction sequence (1) is discussed with special emphasis on the excited energy transfer pathways and the sequence of charge transfer steps: [Formula: see text] where (1)(RC-PC)* denotes the excited singlet state (1)P680* of the reaction centre pigment complex. The structure of the catalytic Mn(4)O(X)Ca cluster of the WOC and the four step reaction sequence leading to oxidative water splitting are described and problems arising for the electronic configuration, in particular for the nature of redox state S(3), are discussed. The unravelling of the mode of O-O bond formation is of key relevance for understanding the mechanism of the process. This problem is not yet solved. A multistate model is proposed for S(3) and the functional role of proton shifts and hydrogen bond network(s) is emphasized. Analogously, the structure of the Q(B) site for PQ reduction to PQH(2) and the energetic and kinetics of the two step redox reaction sequence are described. Furthermore, the relevance of the protein dynamics and the role of water molecules for its flexibility are briefly outlined. We end this review by presenting future perspectives on the water oxidation process.     

Excitation dynamics and heterogeneity of energy equilibration in the core antenna of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Energy equilibration in the photosystem I core antenna from the cyanobacterium Synechocystis sp. PCC 6803 was studied using femtosecond transient absorption spectroscopy at 298 K. The photosystem I core particles were excited at 660, 693, and 710 nm with 150 fs spectrally narrow laser pulses (fwhm = 5 nm). Global analysis revealed three kinetic processes in the core antenna with lifetimes of 250-500 fs, 1.5-2.5 ps, and 20-30 ps. The first two components represent strongly excitation wavelength-dependent energy equilibration processes while the 20-30 ps phase reflects the trapping of energy by the reaction center. Excitation into the blue and red edge of the absorption band induces downhill and uphill energy flows, respectively, between different chlorophyll a spectral forms of the core. Excitation at 660 nm induces a 500 fs downhill equilibration process within the bulk of antenna while the selective excitation of long-wavelength-absorbing chlorophylls at 710 nm results in a 380 fs uphill energy transfer to the chlorophylls absorbing around 695-700 nm, presumably reaction center pigments. The 1.5-2.5 ps phases of downhill and uphill energy transfer are largely equivalent but opposite in direction, indicating energy equilibration between bulk antenna chlorophylls at 685 nm and spectral forms absorbing below 700 nm. Transient absorption spectra with excitation at 693 nm exhibit spectral evolution within approximately 2 ps of uphill energy transfer to major spectral forms at 680 nm and downhill energy transfer to red pigments at 705 nm. The 20-30 ps trapping component and P(700) photooxidation spectra derived from data on the 100 ps scale are largely excitation wavelength independent. An additional decay component of red pigments at 710 nm can be induced either by selective excitation of red pigments or by decreasing the temperature to 264 K. This component may represent one of the phases of energy transfer from inhomogeneously broadened red pigments to P(700). The data are discussed based on the available structural model of the photosystem I reaction center and its core antenna.     

Energy transfer in the major intrinsic light-harvesting complex from Amphidinium carterae

Carbonyl carotenoids are important constituents of the antenna complexes of marine organisms. These carotenoids possess an excited state with a charge-transfer character (intramolecular charge transfer state, ICT), but many details of the carotenoid to chlorophyll energy transfer mechanisms are as yet poorly understood. Here, we employ femtosecond transient absorption spectroscopy to study energy transfer pathways in the intrinsic light-harvesting complex (LHC) of dinoflagellates, which contains the carbonyl carotenoid peridinin. Carotenoid to chlorophyll energy transfer efficiency is about 90% in the 530-550 nm region, where the peridinin S2 state transfers energy with an efficiency of 25-50%. The rest proceeds via the S1/ICT channel, and the major S1/ICT-mediated energy transfer pathway utilizes the relaxed S1/ICT state and occurs with a time constant of 2.6 ps. Below 525 nm, the overall energy transfer efficiency drops because of light absorption by another carotenoid, diadinoxanthin, that contributes only marginally to energy transfer. Instead, its role is likely to be photoprotection. In addition to the peridinin-Chl-a energy transfer, it was shown that energy transfer also occurs between the two chlorophyll species in LHC, Chl-c2, and Chl-a. The time constant characterizing the Chl-c2 to Chl-a energy transfer is 1.4 ps. The results demonstrate that the properties of the S1/ICT state specific for carbonyl carotenoids is the key to ensure the effective harvesting of photons in the 500-600 nm region, which is of vital importance to underwater organisms.     

Investigating the Mechanism of Electron Transfer to the Binuclear Center in Cu-Heme Oxidases

Novel experimental evidence is presented further supporting the hypothesis that, starting with resting oxidized cytochrome c oxidase, the internal electron transfer to the oxygen binding site is kinetically controlled. The reduction of the enzyme was followed spectroscopically and in the presence of NO or CO, used as trapping ligands for reduced cytochrome a₃; ruthenium hexamine was used as a spectroscopically silent electron donor. Consistent with the high combination rate constant for reduced cytochrome a₃, NO proved to be a very efficient trapping ligand, while CO did not. The results are discussed in view of two alternative (thermodynamic and kinetic) hypotheses of control of electron transfer to the binuclear (cyt.a₃-Cu_B) center. Fulfilling the prediction of the kinetic control hypothesis: i) the reduction of cytochrome a₃ and ligation are synchronous and proceed at the intrinsic rate of cytochrome a₃ reduction, ii) the measured rate of formation of the nitrosyl derivative is independent of the concentration of both the reductant and NO.