Initial electron-transfer in the reaction center from Rhodobacter sphaeroides

The initial electron transfer steps in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides have been investigated by femtosecond time-resolved spectroscopy. The experimental data taken at various wavelengths demonstrate the existence of at least four intermediate states within the first nanosecond. The difference spectra of the intermediates and transient photodichroism data are fully consistent with a sequential four-step model of the primary electron transfer: Light absorption by the special pair P leads to the state P*. From the excited primary donor P*, the electron is transferred within 3.5 +/- 0.4 ps to the accessory bacteriochlorophyll B. State P+B- decays with a time constant of 0.9 +/- 0.3 ps passing the electron to the bacteriopheophytin H. Finally, the electron is transferred from H- to the quinone QA within 220 +/- 40 ps.     

Electron transfer in the Rhodobacter sphaeroides reaction center assembled with zinc bacteriochlorophyll

The cofactor composition and electron-transfer kinetics of the reaction center (RC) from a magnesium chelatase (bchD) mutant of Rhodobacter sphaeroides were characterized. In this RC, the special pair (P) and accessory (B) bacteriochlorophyll (BChl) -binding sites contain Zn-BChl rather than BChl a. Spectroscopic measurements reveal that Zn-BChl also occupies the H sites that are normally occupied by bacteriopheophytin in wild type, and at least 1 of these Zn-BChl molecules is involved in electron transfer in intact Zn-RCs with an efficiency of >95% of the wild-type RC. The absorption spectrum of this Zn-containing RC in the near-infrared region associated with P and B is shifted from 865 to 855 nm and from 802 to 794 nm respectively, compared with wild type. The bands of P and B in the visible region are centered at 600 nm, similar to those of wild type, whereas the H-cofactors have a band at 560 nm, which is a spectral signature of monomeric Zn-BChl in organic solvent. The Zn-BChl H-cofactor spectral differences compared with the P and B positions in the visible region are proposed to be due to a difference in the 5th ligand coordinating the Zn. We suggest that this coordination is a key feature of protein–cofactor interactions, which significantly contributes to the redox midpoint potential of H and the formation of the charge-separated state, and provides a unifying explanation for the properties of the primary acceptor in photosystems I (PS1) and II (PS2).     

Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors.

The three-dimensional structure of the cofactors of the reaction center of Rhodobacter sphaeroides R-26 has been determined by x-ray diffraction and refined at a resolution of 2.8 A with an R value of 26%. The main features of the structure are similar to the ones determined for Rhodopseudomonas viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 5, 2445-2451]. The cofactors are arranged along two branches, which are approximately related to each other by a 2-fold symmetry axis. The structure is well suited to produce light-induced charge separation across the membrane. Most of the structural features predicted from physical and biochemical measurements are confirmed by the x-ray structure.     

Structure of the reaction center from Rhodobacter sphaeroides R-26: membrane-protein interactions.

The energetics of membrane-protein interactions are analyzed with the three-dimensional model of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides. The position of the RC in the membrane and the thickness of the membrane were obtained by minimizing the hydrophobic energy with the energy function of Eisenberg and McLachlan. The 2-fold symmetry axis that relates the L and M subunits is, within the accuracy of 5 degrees, parallel to the normal of the membrane. The thickness of the membrane is estimated to be 40-45 A. Residues that are exposed to the membrane are relatively poorly conserved in the sequences of homologous RC proteins. The surface area of the RC is comparable to the surface areas of water-soluble proteins of similar molecular weight. The volumes of interior atoms in the RC are also similar to those of water-soluble proteins, indicating the same compact packing for both types of proteins. The electrostatic potential of the cofactors was calculated. The results show an asymmetry in the potential between the two possible pathways of electron transfer, with the A branch being preferred electrostatically.     

Structure of the reaction center from Rhodobacter sphaeroides R-26: the protein subunits.

The three-dimensional structure of the protein subunits of the reaction center (RC) of Rhodobacter sphaeroides has been determined by x-ray diffraction at a resolution of 2.8 A with an R factor of 26%. The L and M subunits each contain five transmembrane helices and several helices that do not span the membrane. The L and M subunits are related to each other by a 2-fold rotational symmetry axis that is approximately the same as that determined for the cofactors. The H subunit has one transmembrane helix and a globular domain on the cytoplasmic side, which contains a helix that does not span the membrane and several beta-sheets. The structural homology with RCs from other purple bacteria is discussed. A structure of the complex formed between the water soluble cytochrome c2 and the RC from Rb. sphaeroides is proposed.     

Structure of the reaction center from Rhodobacter sphaeroides R-26: protein-cofactor (quinones and Fe2+) interactions.

The three-dimensional structure of the reaction center (RC) from Rhodobacter sphaeroides has been determined by x-ray diffraction to a resolution of 2.8 A with an R value of 24%. The interactions of the protein with the primary quinone, QA, secondary quinone, QB, and the nonheme iron are described and compared to those of RCs from Rhodopseudomonas viridis. Structural differences between the QA and QB environments that contribute to the function of the quinones (the electron transfer from QA- to QB and the charge recombination of QA-, QB- with the primary donor) are delineated. The protein residues that may be involved in the protonation of QB are identified. A pathway for the doubly reduced QB to dissociate from the RC is proposed. The interactions between QB and the residues that have been changed in herbicide-resistant mutants are described. The environment of the nonheme iron is compared to the environments of metal ions in other proteins.     

Structure of the reaction center from Rhodobacter sphaeroides R-26 and 2.4.1: protein-cofactor (bacteriochlorophyll, bacteriopheophytin, and carotenoid) interactions.

The three-dimensional structures of the cofactors and protein subunits of the reaction center (RC) from the carotenoidless mutant strain of Rhodobacter sphaeroides R-26 and the wild-type strain 2.4.1 have been determined by x-ray diffraction to resolutions of 2.8 A and 3.0 A with R values of 24% and 26%, respectively. The bacteriochlorophyll dimer (D), bacteriochlorophyll monomers (B), and bacteriopheophytin monomers (phi) form two branches, A and B, that are approximately related by a twofold symmetry axis. The cofactors are located in hydrophobic environments formed by the L and M subunits. Differences in the cofactor-protein interactions between the A and B cofactors, as well as between the corresponding cofactors of Rb, sphaeroides and Rhodopseudomonas viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 3, 2445-2451], are delineated. The roles of several structural features in the preferential electron transfer along the A branch are discussed. Two bound detergent molecules of beta-octyl glucoside have been located near BA and BB. The environment of the carotenoid, C, that is present in RCs from Rb. sphaeroides 2.4.1 consists largely of aromatic residues of the M subunit. A role of BB in the triplet energy transfer from D to C and the reason for the preferential ease of removal of BB from the RC is proposed.     

Multiple pathways for ultrafast transduction of light energy in the photosynthetic reaction center of Rhodobacter sphaeroides

A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (BA*) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P+HA-. In experiments in which P or BA was selectively excited at 880 nm or 796 nm, respectively, the formation of P+HA- was associated with similar time constants of 1.5 ps and 1. 7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P+BA- and P* is formed in parallel from BA* on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (BB) and the high exciton component of P (P+) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.     

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.     

Protonation of interacting residues in a protein by a Monte Carlo method: application to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides.

We used Monte Carlo methods to treat statistical problem of electrostatic interactions among many titrating amino acids and applied these methods to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides, including all titrating sites. We computed the average protonation of residues as a function of pH from an equilibrium distribution of states generated by random sampling. Electrostatic energies were calculated from a finite difference solution to the linearized Poisson-Boltzmann equation using the coordinates from solved protein structures. For most calculations we used the Metropolis algorithm to sample protonation states; for strongly coupled sites, we substantially reduced sampling errors by using a modified algorithm that allows multiple site transitions. The Monte Carlo method agreed with calculations for a small test system, lysozyme, for which the complete partition function was calculated. We also calculated the pH dependence of the free energy change associated with electron transfer from the primary to the secondary quinone in the photosynthetic reaction center. The shape of the resulting curve agreed fairly well with experiment, but the proton uptake from which the free energy was calculated agreed only to within a factor of two with the observed values. We believe that this discrepancy resulted from errors in the individual electrostatic energy calculations rather than from errors in the Monte Carlo sampling.     

Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides

Photosynthetic bacterial reaction centers convert light excitation into chemical free energy. The initial electron transfer leads to the consecutive semireductions of the primary (Q(A)) and secondary (Q(B)) quinone acceptors. The Q(A)(-) and Q(B)(-) formations induce proton uptake from the bulk. Their magnitudes (H(+)/Q(A)(-) and H(+)/Q(B)(-), respectively) probe the electrostatic interactions within the complex. The pH dependence of H(+)/Q(A)(-) and H(+)/Q(B)(-) were studied in five single mutants modified at the L209 site (L209P-->F,Y,W,E,T). This residue is situated at the border of a continuous chain of water molecules connecting Q(B) to the bulk. In the wild type (WT), a proton uptake band is present at high pH in the H(+)/Q(A)(-) and H(+)/Q(B)(-) curves and is commonly attributed to a cluster of acidic groups situated nearby Q(B). In the H(+)/Q(A)(-) curves of the L209 variants, this band is systematically absent but remains in the H(+)/Q(B)(-) curves. Moreover, notable increase of H(+)/Q(B)(-) is observed in the L209 mutants at neutral pH as compared with the WT. The large effects observed in all L209 mutants are not associated with significant structural changes (Kuglstatter, A., Ermler, U., Michel, H., Baciou, L. & Fritzsch, G. Biochemistry (2001) 40, 4253-4260). Our data suggest that, in the L209 mutants, the Q(B) cluster does not respond to the Q(A)(-) formation as observed in the WT. We propose that, in the mutants, removal of the rigid proline L209 breaks a necessary hydrogen bonding connection between the quinone sites. These findings suggest an important role for structural rigidity in ensuring a functional interaction between quinone binding sites.     

Structure of the reaction center from Rhodobacter sphaeroides R-26 and 2.4.1: symmetry relations and sequence comparisons between different species.

Photosynthetic reaction centers from purple bacteria exhibit an approximate twofold symmetry axis, which relates both the cofactors and the L and M subunits. For the reaction center from Rhodobacter sphaeroides, deviations from this twofold symmetry axis have been quantitated by superposing, by a 180 degrees rotation, the cofactors of the B branch onto the A branch and the M subunit onto the L subunit. An alignment of the sequences of the L and M subunits from four purple bacteria, one green bacterium, and the D1 and D2 subunits of a photosystem II-containing green alga is presented. The residues that are conserved in all six species are shown in relation to the structure of Rb. sphaeroides and their possible role in the function of the reaction center is discussed. A method is presented for characterizing the exposure of alpha-helices to the membrane based on the periodicity of conserved residues. This method may prove useful for modeling the three-dimensional structures of membrane proteins.     

Specific alteration of the oxidation potential of the electron donor in reaction centers from Rhodobacter sphaeroides.

The effects of multiple changes in hydrogen bond interactions between the electron donor, a bacteriochlorophyll dimer, and histidine residues in the reaction center from Rhodobacter sphaeroides have been investigated. Site-directed mutations were designed to add or remove hydrogen bonds between the 2-acetyl groups of the dimer and histidine residues at the symmetry-related sites His-L168 and Phe-M197, and between the 9-keto groups and Leu-L131 and Leu-M160. The addition of a hydrogen bond was correlated with an increase in the dimer midpoint potential. Measurements on double and triple mutants showed that changes in the midpoint potential due to alterations at the individual sites were additive. Midpoint potentials ranging from 410 to 765 mV, compared with 505 mV for wild type, were achieved by various combinations of mutations. The optical absorption spectra of the reaction centers showed relatively minor changes in the position of the donor absorption band, indicating that the addition of hydrogen bonds to histidines primarily destabilized the oxidized state of the donor and had little effect on the excited state relative to the ground state. Despite the change in energy of the charge-separated states by up to 260 meV, the mutant reaction centers were still capable of electron transfer to the primary quinone. The increase in midpoint potential was correlated with an increase in the rate of charge recombination from the primary quinone, and a fit of these data using the Marcus equation indicated that the reorganization energy for this reaction is approximately 400 meV higher than the change in free energy in wild type. The mutants were still capable of photosynthetic growth, although at reduced rates relative to the wild type. These results suggest a role for protein-cofactor interactions--in particular, histidine-donor interactions--in establishing the redox potentials needed for electron transfer in biological systems.     

Pathway of proton transfer in bacterial reaction centers: second-site mutation Asn-M44-->Asp restores electron and proton transfer in reaction centers from the photosynthetically deficient Asp-L213-->Asn mutant of Rhodobacter sphaeroides.

Site-directed mutagenesis of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides has shown Asp-213 of the L subunit (Asp-L213) to be important for photosynthetic viability. Replacement of Asp-L213 with Asn resulted in a photosynthetically deficient mutant, due to the 10(4)-fold slower rate for the proton-coupled electron transfer reaction QA-QB- + 2H+-->QAQBH2 (k(2)AB). The detrimental effect of Asn-L213 is surprising since RCs from Rhodopseudomonas viridis, Rhodospirillum rubrum, and Chloroflexus aurantiacus have Asn at the homologous position. However, RCs from these bacteria have an Asp located near QB (the secondary quinone acceptor) at the position homologous to Asn-M44 in Rb. sphaeroides which might function in place of Asp-L213. To test this conjecture a "viridis-like" structure was introduced into Rb. sphaeroides by replacing Asp-L213 with Asn and Asn-M44 with Asp. The RCs from this double mutant displayed near-native rates for the electron transfer reaction k(2)AB and restored photosynthetic competence. The rates for the first electron transfer reaction QA-QB-->QAQB- (k(1)AB) and charge recombination D+QAQB--->DQAQB (kBD) were also restored to near-native values. These results indicate that Asp at either the L213 or the M44 site near QB can provide a pathway for rapid proton transfer and explain why Asp-L213 need not be conserved in different photosynthetic bacteria. To test further the effect of Asp at M44 on electron and proton transfer to QB a mutant containing Asp at both L213 and M44 was constructed. The RCs from this mutant (Asn-M44-->Asp) exhibited faster proton-coupled electron transfer to QB-. The increased rate of proton-coupled electron transfer (k(2)AB) in the presence of negatively charged Asp residues near QB suggests the role of an Asp near QB as (i) a proton donor group in the proton transfer chain and/or (ii) a negatively charged residue stabilizing proton transfer to reduced QB.     

Femtosecond spectroscopy of electron transfer in the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26: Direct electron transfer from the dimeric bacteriochlorophyll primary donor to the bacteriopheophytin acceptor with a time constant of 2.8 +/- 0.2 psec

The primary light-induced charge separation in reaction centers from Rhodopseudomonas sphaeroides R-26 has been investigated after excitation with laser pulses of 150 fsec duration within the longwave absorption band of the primary donor at 850 nm. An excited state of the primary donor, characterized by a broad absorption spectrum extending over the whole spectral range investigated (545-1240 nm), appeared within 100 fsec and gave rise to stimulated emission in the 870- to 1000-nm region with a 2.8-psec lifetime. The photooxidation of the primary donor, as measured at 1240 nm, and the photoreduction of the bacteriopheophytin acceptor, monitored at 545 nm and 675 nm, have been found to proceed simultaneously with a time constant of 2.8 ± 0.2 psec. Kinetics of absorbance changes at other probe wavelengths gave no indication that an accessory bacteriochlorophyll is involved as a transient electron acceptor.     

Effect of specific mutations of tyrosine-(M)210 on the primary photosynthetic electron-transfer process in Rhodobacter sphaeroides.

We have measured the rate of the initial electron-transfer process as a function of temperature in reaction centers in a native strain of the photosynthetic bacterium Rhodobacter sphaeroides and two mutants generated by site-directed mutagenesis. In the mutants, a tyrosine residue in the vicinity of the primary electron donor and acceptor molecules was replaced by either phenylalanine or isoleucine. The electron-transfer reaction is slower in the mutants and has a qualitatively different dependence on temperature. In native reaction centers the rate increases as the temperature is reduced, in the phenylalanine mutant it is virtually independent of temperature, and in the isoleucine mutant it decreases with decreasing temperature. At 77 K, the electron-transfer reaction is approximately 30 times slower in the isoleucine mutant than in the native. These observations support the view that tyrosine-(M)210 plays an important role in the electron-transfer mechanism. In the isoleucine mutant at low temperatures, the stimulated emission from the excited reaction center undergoes a time-dependent shift to shorter wavelengths.     

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a₃ (Fe⁴⁺ = O²⁻) to the oxidized form (Fe³⁺OH⁻). This redox step requires the delivery of a “chemical” H⁺ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660–669]. The D channel is likely to be involved in the uptake of both “chemical” and “pumped” protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a₃ in the K362M and E286Q mutants under aerobic steady-state turnover conditions.     

From antenna to reaction center: Pathways of ultrafast energy and charge transfer in photosystem II

The photosystem II core complex (PSII-CC) is the smallest subunit of the oxygenic photosynthetic apparatus that contains core antennas and a reaction center, which together allow for rapid energy transfer and charge separation, ultimately leading to efficient solar energy conversion. However, there is a lack of consensus on the interplay between the energy transfer and charge separation dynamics of the core complex. Here, we report the application of two-dimensional electronic-vibrational (2DEV) spectroscopy to the spinach PSII-CC at 77 K. The simultaneous temporal and spectral resolution afforded by 2DEV spectroscopy facilitates the separation and direct assignment of coexisting dynamical processes. Our results show that the dominant dynamics of the PSII-CC are distinct in different excitation energy regions. By separating the excitation regions, we are able to distinguish the intraprotein dynamics and interprotein energy transfer. Additionally, with the improved resolution, we are able to identify the key pigments involved in the pathways, allowing for a direct connection between dynamical and structural information. Specifically, we show that C505 in CP43 and the peripheral chlorophyll Chlz_D1 in the reaction center are most likely responsible for energy transfer from CP43 to the reaction center.

Photosynthesis is the process through which solar energy is converted into chemical energy (1–3). Photosystem II (PSII), a pigment–protein complex found in cyanobacteria, algae, and land plants, is the site of water splitting and is therefore crucial for photosynthetic function (4–6). It is connected with a large light-harvesting antenna system that collects solar energy and transfers the energy to the reaction center (RC), where charge separation (CS) occurs. Unlike the antenna system of purple bacteria that has a clear energy funnel, the PSII antenna system has a more complicated composition and a very complex energy landscape (4–7). These features allow for regulation that responds to rapid environmental fluctuations and protect the organisms in, for example, excess light, while maintaining highly efficient electronic energy transfer (EET) under optimal conditions (8). To understand the intricate interactions between the subunits that allow for the robustness of this photosynthetic system, the first step is to understand how the antenna system is connected to the RC. The PSII core complex (PSII-CC) is the smallest unit in which the RC is connected to the antenna proteins. It is a dimeric pigment–protein complex in which each monomer contains an RC and two core antenna proteins, namely, CP43 and CP47 (1, 7). These core antennas not only harvest solar energy but also act as the crucial bridge between the peripheral light-harvesting antenna system and the RC. Fig. 1A shows the pigment arrangement of the PSII-CC. The RC, consisting of the D1 and D2 branches, binds the following pigments: 1) two special pair chlorophyll a (P_D1 and P_D2), 2) two accessory chlorophyll a (Chl_D1 and Chl_D2), 3) two pheophytin a (Pheo_D1 and Pheo_D2), and 4) two peripheral chlorophyll a (Chlz_D1 and Chlz_D2) (9, 10). Despite the similarity between the D1 and D2 branches, CS occurs only along the D1 branch (11, 12). CP43, one of the two core antenna proteins, contains 13 chlorophyll a (Chls) and is located closer to the D1 active branch. CP47 contains 16 Chls and is located closer to the D2 branch (10). Together, these proteins provide highly effective EET and CS, which are key to the high quantum yield of CS in the RC.Open in a separate windowFig. 1.(A) Pigment arrangement of monomeric PSII-CC (whereas it is typically found as a dimer) depicted based on the cryoelectron microscopy structure (3JCU) reported by Wei et al. (10). The pigments of CP43, RC, and CP47 are shown in green, blue, and red, respectively. (B) Corresponding excitonic energy levels of monomeric PSII-CC color coded to match pigments in A (55–57). The gray shaded regions in the background represent the three groups based on similar characteristic dynamics. Note that the boundaries between the groups provide only a rough separation region as the dynamical behaviors change gradually along ω_exc. The asterisk (for the RC state) indicates an optically dark state.Despite the importance of the PSII-CC, its early time dynamics is not fully understood—specifically the competition between EET and CS (5, 7). This is largely due to the highly congested excitonic manifold (Fig. 1B) and ultrafast EET timescales, which challenge ultrafast spectroscopic techniques. Two distinct models have been put forth to try to describe the function of the PSII-CC. These two models are the “exciton/radical pair equilibrium” (ERPE) model (13–17) and the “transfer-to-trap limited” (TTTL) model (18–22). An early fluorescence decay experiment (13, 14) suggested that rapid EET allows the excitonic states to reach an equilibrium between the core antennas and the RC before CS occurs (k_EET ≫ k_CS), which is the basis for the ERPE model. This model was later supported by improved time-resolved fluorescence (15) and transient absorption experiments (16). However, a major discrepancy in this model arose with the measurement of the X-ray crystal structure of the PSII-CC (18). It was suggested that the large distances (center-to-center distance, >20 Å) between antenna and RC pigments resolved in the crystal structure would mean that ultrafast EET between the antenna proteins and the RC is unlikely. A model was then put forth that instead suggested that the EET from the core antenna to the RC is slow compared to CS (k_EET ≪ k_CS), and therefore, the EET to the trap becomes a kinetic bottleneck (18). This TTTL model was later supported by transient infrared (IR) (19) and time-resolved fluorescence experiments (20, 21) as well as structure-based simulations (22). Additionally, Kaucikas et al. (23) performed a polarized transient IR experiment on an oriented single PSII-CC crystal. The decay of the polarization-dependent signature (50–100 ps) observed in their experiment suggests that equilibration between different subunits is slow, consistent with the TTTL model. However, it has been pointed out that satisfactory fitting of the spectral evolution to this model does not necessarily imply that it is correct (24, 25), especially as others have shown that the EET dynamics cannot be adequately described by a single hopping scheme (26, 27). A recent two-dimensional electronic spectroscopy (2DES) experiment (28) with improved time resolution has also revealed the existence of ultrafast EET (<100 fs) that was not predicted by theoretical calculations. In their work, Pan et al. (28) attributed the origin of this unexpectedly fast EET pathway to polaron formation. Vibronic effects on the ultrafast EET and CS dynamics of other photosynthetic proteins have also been discussed (29–38).The lack of detailed understanding of the PSII-CC early time dynamics, in particular the EET between the core antennas and the RC, highlights the need for further experimental input with the ability to make specific assignments of the dynamical pathways. This, however, requires simultaneous high temporal and spectral resolution, which remains a challenge for ultrafast spectroscopic techniques. Here, we describe the application of two-dimensional electronic-vibrational (2DEV) spectroscopy (39–41) to the PSII-CC. The combination of both spectral dimensions provides an improved resolution that allows us to obtain much more detailed dynamical information in complex systems. The excitonic energy landscapes generated by electronic coupling in photosynthetic complexes, combined with site-dependent and charge state–dependent vibrational spectra, allow the resolution along both axes of 2DEV spectra to provide a direct connection between energetic space (via visible excitation) and physical space (via IR detection). This advantage has proven to be useful for the studies of dynamics in photosynthetic pigment–protein complexes (33, 40–45). Specifically, the resolution along the electronic excitation axis allows for the separation of the contributions from different pathways, while the resolution along the vibrational detection axis provides a way to identify the protein subunits or even specific states involved in the dynamics. As we will show, this unique feature of 2DEV spectroscopy provides insight into the complex dynamics of the PSII-CC.In the following text, we will show that the sub-100-ps dynamics of the PSII-CC extracted from spinach are highly dependent on the excitation frequency range. The resolution along the detection axis allows different dominant dynamics to be identified. In addition, we will demonstrate how 2DEV spectroscopy allows us to connect the observed dynamics to specific excitonic states. This connection allows us to obtain a more specific pigment assignment for the EET pathways and therefore provides a more detailed understanding of the finely tuned interactions between the RC and the core antennas (specifically CP43, which is closer to the active D1 branch). We will conclude with a comparison between our results and the existing models in order to provide a path forward in the understanding of this critical photosynthetic component.     

Conformational gating of the electron transfer reaction QA−⋅QB → QAQB−⋅ in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

The mechanism of the electron transfer reaction, Q_A⁻Q_B → Q_AQ_B⁻_, was studied in isolated reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides by replacing the native Q₁₀ in the Q_A binding site with quinones having different redox potentials. These substitutions are expected to change the intrinsic electron transfer rate by changing the redox free energy (i.e., driving force) for electron transfer without affecting other events that may be associated with the electron transfer (e.g., protein dynamics or protonation). The electron transfer from Q_A⁻ to Q_B was measured by three independent methods: a functional assay involving cytochrome c₂ to measure the rate of Q_A⁻ oxidation, optical kinetic spectroscopy to measure changes in semiquinone absorption, and kinetic near-IR spectroscopy to measure electrochromic shifts that occur in response to electron transfer. The results show that the rate of the observed electron transfer from Q_A⁻ to Q_B does not change as the redox free energy for electron transfer is varied over a range of 150 meV. The strong temperature dependence of the observed rate rules out the possibility that the reaction is activationless. We conclude, therefore, that the independence of the observed rate on the driving force for electron transfer is due to conformational gating, that is, the rate limiting step is a conformational change required before electron transfer. This change is proposed to be the movement, controlled kinetically either by protein dynamics or intermolecular interactions, of Q_B by ≈5 Å as observed in the x-ray studies of Stowell et al. [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E. & Feher, G. (1997) Science 276, 812–816].     

Kinetics of photo-induced electron transfer from high-potential iron-sulfur protein to the photosynthetic reaction center of the purple phototroph Rhodoferax fermentans.

The kinetics of photo-induced electrontransfer from high-potential iron-sulfur protein (HiPIP) to the photosynthetic reaction center (RC) of the purple phototroph Rhodoferarfermentans were studied. The rapid photooxidation of heme c-556 belonging to RC is followed, in the presence of HiPIP, by a slower reduction having a second-order rate constant of 4.8 x 10(7) M(-1) x s(-1). The limiting value of kobs at high HiPIP concentration is 95 s(-1). The amplitude of this slow process decreases with increasing HiPIP concentration. The amplitude of a faster phase, observed at 556 and 425 nm and involving heme c-556 reduction, increases proportionately. The rate constant of this fast phase, determined at 425 and 556 nm, is approximately 3 x 10(5) s(-1). This value is not dependent on HiPIP concentration, indicating that it is related to a first-order process. These observations are interpreted as evidence for the formation of a HiPIP-RC complex prior to the excitation flash, having a dissociation constant of -2.5 microM. The fast phase is absent at high ionic strength, indicating that the complex involves mainly electrostatic interactions. The ionic strength dependence of kobs for the slow phase yields a second-order rate constant at infinite ionic strength of 5.4 x 10(6) M(-1) x s(-1) and an electrostatic interaction energy of -2.1 kcal/mol (1 cal = 4.184 J). We conclude that Rhodoferar fermentans HiPIP is a very effective electron donor to the photosynthetic RC.