Spectroelectrochemical determination of the redox potential of pheophytin a,the primary electron acceptor in photosystem II

Thin-layer cell spectroelectrochemistry, featuring rigorous potential control and rapid redox equilibration within the cell, was used to measure the redox potential E_m(Phe a/Phe a⁻) of pheophytin (Phe) a, the primary electron acceptor in an oxygen-evolving photosystem (PS) II core complex from a thermophilic cyanobacterium Thermosynechococcus elongatus. Interferences from dissolved O₂ and water reductions were minimized by airtight sealing of the sample cell added with dithionite and mercury plating on the gold minigrid working electrode surface, respectively. The result obtained at a physiological pH of 6.5 was E_m(Phe a/Phe a⁻) = −505 ± 6 mV vs. SHE, which is by ≈100 mV more positive than the values measured ≈30 years ago at nonphysiological pH and widely accepted thereafter in the field of photosynthesis research. Using the P680* − Phe a free energy difference, as estimated from kinetic analyses by previous authors, the present result would locate the E_m(P680/P680⁺) value, which is one of the key parameters but still resists direct measurements, at approximately +1,210 mV. In view of these pieces of information, a renewed diagram is proposed for the energetics in PS II.     

Interaction between the intermediary electron acceptor (pheophytin) and a possible plastoquinone-iron complex in photosystem II reaction centers

Photoreduction of the intermediary electron acceptor, pheophytin (Pheo), in photosystem II reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and redox potential E_h = -450 mV produces an EPR doublet centered at g = 2.00 with a splitting of 52 G at 7 K in addition to a narrow signal attributed to Pheo^[unk] (g = 2.0033, ΔH ≈ 13 G). The doublet is eliminated after extraction of lyophilized TSF-II with hexane containing 0.13-0.16% methanol but is restored by reconstitution with plastoquinone A (alone or with β-carotene) although not with vitamin K₁. TSF-II and TSF-IIa are found to contain ≈2 nonheme Fe atoms per reaction center. Incubation with 0.55 M LiClO₄ plus 2.5 mM o-phenanthroline (but not with 0.55 M LiClO₄ alone) decreases this value to ≈0.6 and completely eliminates the EPR doublet, but photoreduction of Pheo is not significantly affected. Partial restoration of the doublet (about 25%) was achieved by subsequent incubation with 0.2 mM Fe²⁺, but not with either Mn²⁺ or Mg²⁺. The Fe removal results in the development of a photoinduced EPR signal (g = 2.0044 ± 0.0003, ΔH = 9.2 ± 0.5 G) at E_h = 50 mV, which is not observed after extraction with 0.16% methanol in hexane. It is ascribed to plastosemiquinone no longer coupled to Fe in photosystem II reaction centers. The results show that a complex of plastoquinone and Fe can act as the stable “primary” electron acceptor in photosystem II reaction centers and that the interaction of its singly reduced form with the reduced intermediary acceptor, Pheo^[unk], is responsible for the EPR doublet.     

Redox potential of the terminal quinone electron acceptor QB in photosystem II reveals the mechanism of electron transfer regulation

Photosystem II (PSII) extracts electrons from water at a Mn₄CaO₅ cluster using light energy and then transfers them to two plastoquinones, the primary quinone electron acceptor Q_A and the secondary quinone electron acceptor Q_B. This forward electron transfer is an essential process in light energy conversion. Meanwhile, backward electron transfer is also significant in photoprotection of PSII proteins. Modulation of the redox potential (E_m) gap of Q_A and Q_B mainly regulates the forward and backward electron transfers in PSII. However, the full scheme of electron transfer regulation remains unresolved due to the unknown E_m value of Q_B. Here, for the first time (to our knowledge), the E_m value of Q_B reduction was measured directly using spectroelectrochemistry in combination with light-induced Fourier transform infrared difference spectroscopy. The E_m(Q_B⁻/Q_B) was determined to be approximately +90 mV and was virtually unaffected by depletion of the Mn₄CaO₅ cluster. This insensitivity of E_m(Q_B⁻/Q_B), in combination with the known large upshift of E_m(Q_A⁻/Q_A), explains the mechanism of PSII photoprotection with an impaired Mn₄CaO₅ cluster, in which a large decrease in the E_m gap between Q_A and Q_B promotes rapid charge recombination via Q_A⁻.In oxygenic photosynthesis in plants and cyanobacteria, photosystem II (PSII) has an important function in light-driven water oxidation, a process that leads to the generation of electrons and protons for CO₂ reduction and ATP synthesis, respectively (1–3). Photosynthetic water oxidation also produces molecular oxygen as a byproduct, which is the source of atmospheric oxygen and sustains virtually all life on Earth. PSII reactions are initiated by light-induced charge separation between a chlorophyll (Chl) dimer (P680) and a pheophytin (Pheo) electron acceptor, leading to the formation of a P680⁺Pheo⁻ radical pair (4, 5). An electron hole on P680⁺ is transferred to a Mn₄CaO₅ cluster, the catalytic center of water oxidation, via the redox-active tyrosine, Y_Z (D1-Tyr161). At the Mn₄CaO₅ cluster, water oxidation proceeds through a cycle of five intermediates denoted S_n states (n = 0–4) (6, 7). On the electron acceptor side, the electron is transferred from Pheo⁻ to the primary quinone electron acceptor Q_A and then to the secondary quinone electron acceptor Q_B (8, 9). Q_A and Q_B have many similarities: they consist of plastoquinone (PQ), are located symmetrically around a nonheme iron center, and interact with D2 and D1 proteins, respectively, in a similar manner (Fig. 1) (10, 11). However, they play significantly different roles in PSII (8, 9). Q_A is only singly reduced to transfer an electron to Q_B, whereas Q_B accepts one or two electrons. When Q_B is doubly reduced, the resultant Q_B²⁻ takes up two protons to form plastoquinol (PQH₂), which is then released into thylakoid membranes. Differences between Q_A and Q_B could be caused by differences in the molecular interactions of PQ with surrounding proteins in Q_A and Q_B pockets, although the detailed mechanism remains to be clarified (12, 13).Open in a separate windowFig. 1.Redox cofactors in PSII and the electron transfer pathway (blue arrows). For the PSII structure, the X-ray crystallographic structure at 1.9-Å resolution (Protein Data Bank ID code 3ARC) (9) was used. The electron acceptor side is expanded, showing the arrangements of Q_A, Q_B, and the nonheme iron with their molecular interactions. Nearby carboxylic groups are also shown.Electron transfer reactions in PSII are highly regulated by the spatial localization of redox components and their redox potentials (E_m values). Both forward and backward electron transfers are important; backward electron transfers control charge recombination in PSII, and this serves as photoprotection for PSII proteins (5, 14–17). PSII involves specific mechanisms to regulate forward and backward electron transfer reactions in response to environmental changes. For instance, in strong light, some species of cyanobacteria increase the E_m of Pheo to facilitate charge recombination. Specifically, they exchange D1 subunits originating from different psbA genes to change the hydrogen bond interactions of Pheo (16–20). On the other hand, it was found that impairment of the Mn₄CaO₅ cluster led to a significant increase in the E_m of Q_A by ∼150 mV (21–27). This potential increase was thought to inhibit forward electron transfer to Q_B to promote direct relaxation of Q_A⁻ without forming triplet-state Chl, a precursor of harmful singlet oxygen (2, 5, 14, 15, 17, 23). In addition, charge recombination of Q_A⁻ with P680⁺ prevents oxidative damage by high-potential P680⁺ (5). However, the full mechanism of photoprotection by the regulation of the quinone electron acceptor E_m values remains to be resolved, because the E_m of Q_B has not been determined conclusively, and the effect of Mn₄CaO₅ cluster inactivation on it has not been examined (5).Although the E_m of the single reduction of Q_B has been estimated to be ∼80 mV higher than that of Q_A from kinetic and thermodynamic data (28–32), so far no reports have measured the E_m of Q_B directly. In contrast, the E_m of Q_A was measured extensively using chemical or electrochemical titrations and determined to be approximately −100 mV for oxygen-evolving PSII (21–27). The main reason for this difference is due to the fact that the Q_A reaction can be monitored readily by fluorescence measurement in that an increase in fluorescence indicates Q_A⁻ formation (8, 33–35). However, the fluorescence method cannot be used easily to monitor Q_B reduction. Although UV-Vis absorption and electron spin resonance have also been used to monitor Q_A⁻ in redox titration (summarized in ref. 22), so far these methods have not been used to monitor the titration of Q_B, likely because Q_A⁻ and Q_B⁻ give similar signals (36–39). Another spectroscopic method that can be used to monitor Q_A and Q_B reactions is Fourier transform infrared (FTIR) difference spectroscopy, which detects reaction-induced changes in the molecular vibrations of a cofactor and its environment in proteins (40–45). It was previously shown that comparison of FTIR difference spectra upon Q_A⁻ and Q_B⁻ formation showed some characteristic differences in spectral features (46). In particular, bands at 1,721 and 1,745 cm⁻¹, which were assigned to ester C=O vibrations of nearby Pheo molecules affected by the reduction of Q_A and Q_B, respectively, were suggested to be good markers for discriminating between Q_A and Q_B reactions (46).In this study, we directly measured the E_m of Q_B in PSII using spectroelectrochemistry and light-induced FTIR difference spectroscopy. The effect of Mn₄CaO₅ cluster depletion on the E_m value was also examined. Spectroelectrochemistry has been used to accurately measure the E_m values of cofactors in various redox proteins (47, 48) including redox cofactors in PSII (24, 25, 49, 50). FTIR spectroelectrochemistry, which has the additional merit of being able to provide structural information, has also been used to investigate redox reactions of biomolecules and proteins (48, 50–54). This method was recently applied to the nonheme iron center of PSII to examine the effect of Mn depletion on the E_m value and obtain structural information around the nonheme iron (50). The results in our study showed that the E_m of the first reduction of Q_B [E_m(Q_B⁻/Q_B)] was much higher than previously estimated, and the E_m of the second reduction [E_m(PQH₂/Q_B⁻)] was higher than the first reduction. Furthermore, we showed that Mn depletion hardly affected the E_m values of Q_B, in contrast to the large change in the E_m of Q_A (21–27). With these results, the mechanism of photoprotection of PSII when the Mn₄CaO₅ cluster is inactivated is now clearly explained.     

Independent initiation of primary electron transfer in the two branches of the photosystem I reaction center

Photosystem I (PSI) is a large pigment-protein complex that unites a reaction center (RC) at the core with ∼100 core antenna chlorophylls surrounding it. The RC is composed of two cofactor branches related by a pseudo-C2 symmetry axis. The ultimate electron donor, P₇₀₀ (a pair of chlorophylls), and the tertiary acceptor, F_X (a Fe₄S₄ cluster), are both located on this axis, while each of the two branches is made up of a pair of chlorophylls (ec2 and ec3) and a phylloquinone (PhQ). Based on the observed biphasic reduction of F_X, it has been suggested that both branches in PSI are competent for electron transfer (ET), but the nature and rate of the initial electron transfer steps have not been established. We report an ultrafast transient absorption study of Chlamydomonas reinhardtii mutants in which specific amino acids donating H-bonds to the 13¹-keto oxygen of either ec3_A (PsaA-Tyr696) or ec3_B (PsaB-Tyr676) are converted to Phe, thus breaking the H-bond to a specific ec3 cofactor. We find that the rate of primary charge separation (CS) is lowered in both mutants, providing direct evidence that the primary ET event can be initiated independently in each branch. Furthermore, the data provide further support for the previously published model in which the initial CS event occurs within an ec2/ec3 pair, generating a primary ec2⁺ec3^- radical pair, followed by rapid reduction by P₇₀₀ in the second ET step. A unique kinetic modeling approach allows estimation of the individual ET rates within the two cofactor branches.     

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.     

Initial electron donor and acceptor in isolated Photosystem II reaction centers identified with femtosecond mid-IR spectroscopy

Despite the apparent similarity between the plant Photosystem II reaction center (RC) and its purple bacterial counterpart, we show in this work that the mechanism of charge separation is very different for the two photosynthetic RCs. By using femtosecond visible-pump-mid-infrared probe spectroscopy in the region of the chlorophyll ester and keto modes, between 1,775 and 1,585 cm(-1), with 150-fs time resolution, we show that the reduction of pheophytin occurs on a 0.6- to 0.8-ps time scale, whereas P+, the precursor state for water oxidation, is formed after approximately 6 ps. We conclude therefore that in the Photosystem II RC the primary charge separation occurs between the "accessory chlorophyll" Chl(D1) and the pheophytin on the so-called active branch.     

Directed mutations affecting spectroscopic and electron transfer properties of the primary donor in the photosynthetic reaction center

Oligonucleotide-mediated mutagenesis has been used to change the histidine residues that act as axial ligands to the central Mg²⁺ ions of the “special pair” bacteriochlorophylls in the reaction center of Rhodobacter capsulatus. Histidine-173 of the L subunit has been replaced with glutamine, while histidine-200 of the M subunit has been replaced with glutamine, leucine, or phenylalanine. When leucine or phenylalanine is introduced at M200, one of the special pair bacteriochlorophylls is converted to bacteriopheophytin, which generates a heterodimer at the special pair binding site. The pigment composition of the reaction center is unaltered when either histidine is replaced with glutamine. All of these mutant reaction centers are photochemically active, although the electron transfer properties of heterodimer-containing reaction centers are altered. These mutations begin to define the structural parameters that determine whether bacteriochlorophyll or bacteriopheophytin will be incorporated into the tetrapyrrole binding sites of the photosynthetic reaction center. Our results demonstrate that the properties of the photosynthetic reaction center can be changed by directed mutagenesis, which makes this complex an excellent model for testing theories of electron transfer in biological systems.     

Engineering of an alternative electron transfer path in photosystem II

The initial steps of oxygenic photosynthetic electron transfer occur within photosystem II, an intricate pigment/protein transmembrane complex. Light-driven electron transfer occurs within a multistep pathway that is efficiently insulated from competing electron transfer pathways. The heart of the electron transfer system, composed of six linearly coupled redox active cofactors that enable electron transfer from water to the secondary quinone acceptor Q_B, is mainly embedded within two proteins called D1 and D2. We have identified a site in silico, poised in the vicinity of the Q_A intermediate quinone acceptor, which could serve as a potential binding site for redox active proteins. Here we show that modification of Lysine 238 of the D1 protein to glutamic acid (Glu) in the cyanobacterium Synechocystis sp. PCC 6803, results in a strain that grows photautotrophically. The Glu thylakoid membranes are able to perform light-dependent reduction of exogenous cytochrome c with water as the electron donor. Cytochrome c photoreduction by the Glu mutant was also shown to significantly protect the D1 protein from photodamage when isolated thylakoid membranes were illuminated. We have therefore engineered a novel electron transfer pathway from water to a soluble protein electron carrier without harming the normal function of photosystem II.     

Photochemistry and spectroscopy of a five-chlorophyll reaction center of photosystem II isolated by using a Cu affinity column

A reaction center of photosystem II was isolated from Pisum sativum by using immobilized metal affinity chromatography. This reaction center is photochemically active and has a room temperature Qgamma chlorophyll (Chl) absorption band peaking at 677.5 nm. From HPLC analysis, the pigment stoichiometry was suggested to be 5 Chls per 1 beta-carotene per 2 pheophytins. Low-temperature absorption measurements at 77 K were consistent with the removal of one of the Chls associated with the usual form of the reaction center isolated by using ion-exchange chromatography. Transient absorption spectroscopy on the picosecond time scale indicated that the Chl removed belongs to a pool of Chl absorbing at approximately 670 nm (C670II) that transfers energy relatively slowly to the primary donor P680 in support of our recently proposed model. The results also support the previous conclusion that radical pair formation is largely associated with a 21-ps time constant when P680 is directly excited and that the identity of C670II is likely to be peripherally bound Chls possibly ligated to conserved His residues at positions 118 on the D1 and D2 proteins.     

Determination of the primary charge separation rate in isolated photosystem II reaction centers with 500-fs time resolution

We have measured directly the rate of formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a^- (Pheoa^-) following excitation of isolated spinach photosystem II reaction centers at 4°C. The reaction-center complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure that stabilizes the protein complex. Transient absorption difference spectra were measured from 440 to 850 nm as a function of time with 500-fs resolution following 610-nm laser excitation. The formation of P680⁺-Pheoa^- is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 505 and 540 nm due to formation of Pheoa^-. The appearance of the 820-nm band is monoexponential with τ = 3.0 ± 0.6 ps. The time constant for decay of ^1*P680, the lowest excited singlet state of P680, monitored at 650 nm, is τ = 2.6 ± 0.6 ps and agrees with that of the appearance of P680⁺ within experimental error. Treatment of the photosystem II reaction centers with sodium dithionite and methyl viologen followed by exposure to laser excitation, conditions known to result in accumulation of Pheoa^-, results in formation of a transient absorption spectrum due to ^1*P680. We find no evidence for an electron acceptor that precedes the formation of Pheoa^-.     

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.     

Chlorophyll a dimer: a possible primary electron donor for the photosystem II

In the photosynthetic membrane, there is a particular aggregated state for the chlorophyll a (Chl a) molecules with a specific arrangement responsible for the high efficiency of energy conversion. Chl a monolayers, transferred onto solid substrates, are systems that potentially can mimic the packing of the in vivo system. The association of Chl a in the monolayer results in the formation of dimers with an average size of 3.00 +/- 0.15 nm. Considering the organization of the dimers, we assume that P680 is a dimer with the (anti) parallel transition moments of the constituent. The Chl a macrocycles most likely are tilted to each other by 30 degrees with respect to the membrane plane.     

C-terminal processing of reaction center protein D1 is essential for the function and assembly of photosystem II in Arabidopsis

Photosystem II (PSII) reaction center protein D1 is synthesized as a precursor (pD1) with a short C-terminal extension. The pD1 is processed to mature D1 by carboxyl-terminal peptidase A to remove the C-terminal extension and form active protein. Here we report functional characterization of the Arabidopsis gene encoding D1 C-terminal processing enzyme (AtCtpA) in the chloroplast thylakoid lumen. Recombinant AtCtpA converted pD1 to mature D1 and a mutant lacking AtCtpA retained all D1 in precursor form, confirming that AtCtpA is solely responsible for processing. As with cyanobacterial ctpa, a knockout Arabidopsis atctpa mutant was lethal under normal growth conditions but was viable with sucrose under low-light conditions. Viable plants, however, showed deficiencies in PSII and thylakoid stacking. Surprisingly, unlike its cyanobacterial counterpart, the Arabidopsis mutant retained both monomer and dimer forms of the PSII complexes that, although nonfunctional, contained both the core and extrinsic subunits. This mutant was also essentially devoid of PSII supercomplexes, providing an unexpected link between D1 maturation and supercomplex assembly. A knock-down mutant expressing about 2% wild-type level of AtCtpA showed normal growth under low light but was stunted and accumulated pD1 under high light, indicative of delayed C-terminal processing. Although demonstrating the functional significance of C-terminal D1 processing in PSII biogenesis, our study reveals an unsuspected link between D1 maturation and PSII supercomplex assembly in land plants, opening an avenue for exploring the mechanism for the association of light-harvesting complexes with the PSII core complexes.Photosystem II (PSII) consists of more than 20 subunits. Assembly of this photosystem is a multistep process that functions in a highly coordinated fashion (1–3). The process starts with PSII initiation complexes (D2, PsbE, PsbF, and PsbI), and then D1 and CP47 are sequentially recruited to form CP47-RC complexes, followed by addition of PsbH, PsbM, PsbTc, and PsbR subunits. Next, CP43 and other subunits are added to generate PSII monomers, which develop into PSII dimers. Finally, light-harvesting complex (LHC) II is attached to form PSII supercomplexes. The D1 protein of PSII is prone to photodamage under excessive light conditions (4). To sustain photosynthesis, damaged D1 protein is degraded and replaced with a newly synthesized copy via PSII repair—a highly complex and critical process whose mechanism remains unclear (3, 4).In most oxygen-evolving photosynthetic organisms, D1 protein is synthesized as a precursor (pD1) with a C-terminal tail. The pD1 protein is integrated into the thylakoid membrane and forms the initial PSII reaction center combined with other PSII subunits. The C-terminal tail of pD1 must be cleaved by an endopeptidase named the carboxyl terminal peptidase (Ctp) to produce mature D1, the functional form (5). In the cyanobacterium Synechocystis PCC 6803, there are three Ctp homologs (CtpA, CtpB, and CtpC), but only one, CtpA, is responsible for cleavage of the pD1 C-terminal extension (5). Disruption of CtpA leads to a loss of PSII activity and oxygen evolution from failure to form the manganese cluster (4, 6). The processing of pD1 is also critical for the association of extrinsic proteins on the luminal side to stabilize the PSII complexes (6, 7).In contrast to cyanobacteria, our knowledge of the significance of Ctp enzymes and D1 C-terminal processing is limited in land plants. Previous researchers reported the purification of CtpA-like protein from pea (8) and spinach (9). The spinach study further showed that the recombinant Ctp protein expressed from Escherichia coli displays activity against pD1 (9). However, because we lack a genetic approach, the functional significance of CtpA and C-terminal processing remains unknown in those and other land plants. In this study, we applied genetics to identify a gene (At4g17740) encoding a CtpA enzyme in Arabidopsis and showed that it is required for PSII function and chloroplast development. We found that Arabidopsis CtpA is essential for assembling functional PSII core complexes, dimers, and PSII supercomplexes. The enzyme is also critical for the PSII damage–repair cycle during the photoinhibition process.     

Femtosecond photodichroism studies of isolated photosystem II reaction centers.

Photosynthetic conversion of light energy into chemical potential begins in reaction center protein complexes, where rapid charge separation occurs with nearly unit quantum efficiency. Primary charge separation was studied in isolated photosystem II reaction centers from spinach containing 6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559, and 2 beta-carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution and with the pump laser polarized parallel, perpendicular, and at the magic angle (54.7 degrees) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo- were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Qy transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.     

Subpicosecond equilibration of excitation energy in isolated photosystem II reaction centers.

Photosystem II reaction centers have been studied by femtosecond transient absorption spectroscopy. We demonstrate that it is possible to achieve good photoselectivity between the primary electron donor P680 and the majority of the accessory chlorins. Energy transfer can be observed in both directions between P680 and these accessory chlorins depending on which is initially excited. After excitation of either P680 or the other chlorins, the excitation energy is observed to equilibrate between the majority of these pigments at a rate of 100 +/- 50 fs-1. This energy-transfer equilibration takes place before any electron-transfer reactions and must therefore be taken into account in studies of primary electron-transfer reactions in photosystem II. We also show further evidence that the initially excited P680 excited singlet state is delocalized over at least two chlorins and that this delocalization lasts for at least 200 fs.     

Labeling quinone-binding sites in photosynthetic reaction centers: A 38-kilodalton protein associated with the acceptor side of photosystem II

2-Acetoxymethyl-1,4-naphthoquinone (2-AcOMeNQ) binds with rapid kinetics and high affinity to the primary quinone Q_A site of reaction centers from Rhodopseudomonas capsulata. Binding of 2-AcOMeNQ fully restores electron-transfer activity with kinetics that is similar, but not identical, to that seen with ubiquinone-50. When bound at the Q_A site, 2-AcOMeNQ preferentially labels the L subunit. This preference suggests that 2-AcOMeNQ labels primarily the region of a quinone-binding site that is close to the first isoprenoid unit of the side chain, which is expected from the location and structure of the reaction region of the molecule. In photosystem II particles from Synechococcus sp., 2-AcOMeNQ primarily labels two polypeptides with apparent molecular masses of 38 and 19 kDa. Labeling of only the 38-kDa polypeptide is sufficiently sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to conclude that it is involved in binding quinones on the acceptor side of photosystem II. Although we have not yet identified the 38-kDa protein, its properties suggest that it is the D2 protein. From the DCMU-sensitive labeling and from homologies to functionally important regions of the bacterial reaction-center subunits, we propose that the 38-kDa protein is intimately involved in binding the cofactors that mediate primary photochemistry.     

Photosynthetic electron transport in genetically altered photosystem II reaction centers of chloroplasts.

Using a cotransformation system to identify chloroplast transformants in Chlamydomonas reinhardtii, we converted histidine-195 of the photosystem II reaction center D1 protein to a tyrosine residue. The mutants were characterized by a reduced quantum efficiency for photosynthetic oxygen evolution, which varied in a pH-dependent manner, a reduced capacity to oxidize artificial donors to photosystem II, and P680+ reduction kinetics (microsecond) that were essentially similar to wild type. In addition, a dark-stable radical was detected by ESR in mutant photosystem II particles but not in wild-type particles. This radical was similar in g value and lineshape to chlorophyll or carotenoid cations but could have arisen from a tyrosine-195 cation. The ability of the photosystem II trap (P680+) to oxidize tyrosine residues suggests that the mutant tyrosine residue could be used as a redox-sensitive probe to investigate the environment around the photosystem II trap.     

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.     

Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: wild-type and mutant strains deficient in photosystem II reaction center.

Unstacked thylakoid membrane vesicles were obtained from a homogenate of Chlamydomonas reinhardtii by flotation in a 1.8 M sucrose layer containing 5 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid)-10 mM EDTA (pH 7.5). Sodium dodecyl sulfate-gradient gel electrophoresis showed that the wildtype membranes have a total of at least 33 polypeptides ranging in molecular weights from 68,000 to less than 10,000. The wild-type and three non-photosynthetic mutant strains were studied with respect to their photosynthetic electron transport properties, their fluorescence rise kinetics, and their membrane polypeptide compositions. The results showed a strong correlation between the presence of a membrane polypeptide (molecular weight = 47,000) and the activity of the photosystem II reaction center. This polypeptide is missing from F34 (a mendelian mutant lacking Q, the primary electron acceptor of photosystem II), but is partially restored in a suppressed strain of F34 in which there is an incomplete recovery of photosystem II activity. In a thermosensitive mutant, T4, the same polypeptide is present in reduced amount only in cells grown at 35 degrees but not in those grown at 25 degrees. Evidence from fluorescence rise kinetics and partial photochemical reactions show that the cells grown at 25 degree are similar to wild-type cells but the cells grown at 35 degrees are greatly deficient in Q.     

Redox potentials of primary electron acceptor quinone molecule (QA)- and conserved energetics of photosystem II in cyanobacteria with chlorophyll a and chlorophyll d

In a previous study, we measured the redox potential of the primary electron acceptor pheophytin (Phe) a of photosystem (PS) II in the chlorophyll d-dominated cyanobacterium Acaryochloris marina and a chlorophyll a-containing cyanobacterium, Synechocystis. We obtained the midpoint redox potential (E(m)) values of -478 mV for A. marina and -536 mV for Synechocystis. In this study, we measured the redox potentials of the primary electron acceptor quinone molecule (Q(A)), i.e., E(m)(Q(A)/Q(A)(-)), of PS II and the energy difference between [P680·Phe a(-)·Q(A)] and [P680·Phe a·Q(A)(-)], i.e., ΔG(PhQ). The E(m)(Q(A)/Q(A)(-)) of A. marina was determined to be +64 mV without the Mn cluster and was estimated to be -66 to -86 mV with a Mn-depletion shift (130-150 mV), as observed with other organisms. The E(m)(Phe a/Phe a(-)) in Synechocystis was measured to be -525 mV with the Mn cluster, which is consistent with our previous report. The Mn-depleted downshift of the potential was measured to be approximately -77 mV in Synechocystis, and this value was applied to A. marina (-478 mV); the E(m)(Phe a/Phe a(-)) was estimated to be approximately -401 mV. These values gave rise to a ΔG(PhQ) of -325 mV for A. marina and -383 mV for Synechocystis. In the two cyanobacteria, the energetics in PS II were conserved, even though the potentials of Q(A)(-) and Phe a(-) were relatively shifted depending on the special pair, indicating a common strategy for electron transfer in oxygenic photosynthetic organisms.