Directionality of Electron Transfer in Type I Reaction Center Proteins

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Directionality of Electron Transfer in Type I Reaction Center Proteins: High Frequency EPR Study of PS I with Removed Iron-Sulfur Centers Oleg G. Poluektov, and Lisa Marie Utschig J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b04063 • Publication Date (Web): 01 Jun 2015 Downloaded from http://pubs.acs.org on June 17, 2015

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The Journal of Physical Chemistry

Directionality of Electron Transfer in Type I Reaction Center Proteins: High Frequency EPR Study of PS I with Removed Iron-Sulfur Centers Oleg G. Poluektov and Lisa M. Utschig Chemical Sciences and Engineering Department Argonne National Laboratory Argonne, IL 60439 Abstract. A key step of photosynthetic solar energy conversion involves rapid light-induced sequential electron transfer steps that result in the formation of a stabilized charge separated state. These primary reactions take place in large integral membrane reaction center (RC) proteins wherein a series of donor/acceptor cofactors are specifically positioned for efficient electron transfer. RCs can be divided in two classes, Type I and Type II and examples of both types, photosystem I (PS I) and photosystem II (PS II), are involved in the oxygenic photosynthesis of higher plants, cyanobacteria, and algae. High-resolution X-ray crystal structures reveal that PS I and PS II contain two nearly symmetric branches of redox cofactors, termed the A- and B-branches. While unidirectional ET along the A-branch in Type II RCs is well established, there is still a debate whether or not primary photochemistry in Type I RCs is unidirectional along the A-branch or bidirectional proceeding down both the A- and B-branches. Light-induced electron transfer thru the B-branch has been observed in genetically modified PS I and in native PS I pretreated with strong reducing conditions to reduce three [4Fe-4S] clusters, the terminal electron acceptors of PS I. However, the extent of asymmetry of ET along both cofactor branches remains an open question. To prove that bidirectional ET in PSI is not simply an artifact of a reducing environment or genetic modification and to determine the degree of PSI ET asymmetry, we have examined biochemically modified Synechococcus leopoliensis PSI RCs wherein the [4Fe-4S] clusters FX, FA and FB have been removed to prevent secondary ET from phylloquinones (A1A/A1B) to FX. For these Fe-removed proteins, we observe that ET along both the A- and B-branches occurs with a ratio close to 1. Together with previously reported data, the concomitant structural and kinetic information obtained with HF EPR unambiguously proves the bidirectional nature of ET in PS I over a broad temperature range.

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Introduction. Fossil fuels are the current predominant energy source used world-wide. 1 Due to the increasing demand for energy, new renewable and clean energy sources are needed to supplement, and eventually replace, the environmentally harmful and finite supply of fossil fuels. Solar energy provides one of the most promising sources of energy to meet future energy needs. 2-3 Two possible pathways convert solar energy to useful forms of energy. The first approach involves “solar to electricity” conversion which is realized in man-made photovoltaic devices. The second, a “solar to fuel” approach, stores captured solar energy in high energy chemical bonds of molecules such as H2. This approach is inspired by Nature’s photosynthetic solar energy conversion processes which create a fuel, carbohydrates, from sunlight and water.4 5 Both approaches require efficient light-harvesting and light-induced charge separation (CS) steps. In natural photosynthesis, light-induced long-lived charge separation occurs with a quantum yield that approaches 100%. This efficiency so far is unmatched by any man-made artificial system.6 Thus, fundamental studies aimed at resolving mechanisms of photochemical energy conversion in natural photosynthetic systems will provide key insight about design features for both “solar to fuel” and “solar to electricity” bio-inspired energy conversion systems. The initial photosynthetic energy conversion reactions take place in large integral membrane pigment protein complexes known as reaction centers (RCs).4 Charge separation is accomplished via a sequence of rapid electron transfer (ET) reactions initiated from a photoexcited electron donor to a series of redox-active cofactors. All known RCs can be divided in two classes, Type I and Type II. Photosystem I (PS I) and photosystem II (PS II) are the core representatives of these broad classes of photosynthetic proteins. High-resolution X-ray crystal structures reveal that both PS I and PS II contain two nearly symmetrical chains of redox cofactors that extend across the membrane (Figure 1). 7-10 Both of these branches (termed A and B) could potentially participate in the electron transfer (ET) reactions that result in charge separation across the membrane. Significant questions about the charge separation process in the RCs address both the direction of ET and the control of directionality; i.e. does ET occur along one (unidirectional) or both (bidirectional) of the nearly equivalent potential electron transfer pathways and what features of the protein environments influence ET pathways? The unidirectional nature of ET in Type II RCs is well established with light-driven primary ET taking place exclusively through the A-branch of redox cofactors.11 Primary photoinitiated ET starts from excited states of the primary donor P (an electronically coupled pair of chlorophyll) to an intermediate electron acceptor IA (a pheophytin molecule) and then to the quinone primary acceptor QA (Figure 1a). This results in formation of a metastable chargeseparated state, P+QA-. Subsequently, the electron reaches the final quinone acceptor, QB, located in the B-branch. Following two successive turnovers of RC photochemistry the hydroquinone QBH2 leaves the protein for further chemical transformation. Thus the


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unidirectional ET in Type II RCs along A-branch can be rationalized by the different function of the quinone molecules QA and QB. By analogy with Type II RCs, the ET in Type I RCs was assumed to be unidirectional as well. However, the function of quinones in PS I is quite different than that of the quinones in Type II RCs; neither quinone (A1A/A1B) is a terminal acceptor nor leaves the protein upon reduction.12 Instead, following the primary photo-induced reduction of the quinone, A1, the electron is transferred thru a sequence of three [4Fe-4S] clusters termed FX, FA, and FB (Figure 1b). Therefore, different functionality of terminal acceptor molecules cannot be a rational to explain unidirectional ET in PS I. The question of whether or not primary photochemistry proceeds down both cofactor branches, A and B, in Type I RCs was debated for a long time. 13-19 Using advanced high frequency (HF), time-resolved (TR) EPR methods, we were able to directly observe two distinct transient spectra of spin-correlated radical pairs P+A1- from a cyanobacterial PSI RC protein.20 We demonstrated that the geometries of the two distinct donor/acceptor pairs correspond to the charge-separated states along the A- and B-branches, and that our assignments of radical pair geometries are in excellent agreement with the X-ray crystal structure of PS I, thus confirming the bidirectional ET in PS I (Figure 1b). Note that, in these EPR experiments, ET along B-branch in PSI was observed at low temperature (100 K) and under strongly reducing conditions. This definitive observation of bidirectionality was later confirmed by a number of mutagenesis studies of Type I RCs.21-28 In mutagenesis experiments, one of the ET pathways is shut by altering the Figure 1. Schematic structure and ET pathways in Type II (A), Type I (B), and Type I down with iron-sulfur complexes removed (C). The donor-acceptor cofactors in PS II (Type II) protein/cofactor structure, and PS I (Type I) photosynthetic RCs are arranged in two symmetric branches, A (red) which leads to redirection of and B (blue). A. In Type II RC from Rhodobactor sphaeroides the primary donor P is a pair of the ET from one branch to the bacteriochlorophyll molecules, intermediate acceptors IA/B - bacteriopheophytin, and terminal acceptors QA/B – ubiquinones positioned around a non-heme Fe ion. other branch. This approach Unidirectional ET pathway is indicated by arrows. After a two-electron, two-proton makes it impossible to reduction QBH2 is released from the RC, transporting electrons and protons to other redox determine the extent of components in the bacteria. B. In Type I RCs the terminal electron acceptors are Fe-S clusters. In PS I, following asymmetry of ET along Aphotoexcitation, the primary donor P becomes oxidized, transferring its electron to one of two identical chains of donor/acceptor molecules: a chlorophyll A0, phylloquinone A1, and B-branches. The same is and three [4Fe-4S] clusters, FX, FA, and FB. Photoinduced ET in PSI is bidirectional, true for strongly reduced proceeding through both the A- and B-branches of cofactors.20 C. Type I RC with iron-sulfur complexes removed. ET cannot proceed past the quinones samples. To prove that the A1A/B.



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reduction condition of the sample preparation does not influence our conclusion of bidirectional ET in PS I and to clarify the degree of the ET asymmetry, herein we report TR HF EPR (D-band, 130 GHz) measurements of light-induced CS on modified PSI RCs wherein the acceptor ironsulfur centers FX, FA and FB have been removed to prevent secondary forward ET from phylloquinones A1 to the iron-sulfur complex FX (Figure 1c). Comparative analysis of HF TR EPR data obtained for RCs with either removed or reduced iron-sulfur complexes clearly demonstrate that, even at low temperatures, the ET is bidirectional and the ratio of ET through A and B branches is close to 1.

Experimental Sample preparation PSI reaction centers were extracted from 99% deuterated cyanobacterium Synechococcus leopoliensis 29 as described in 30 Purified PSI was prepared in 50 mM MES, pH 6.53, 20 % glycerol, 0.03% β-DM (n-dodecyl β-D-maltopyranoside, Anatrace), 30 mM sodium ascorbate. The Fe-S clusters were removed by established preparations. PSI (0.2 mg chl/ml) was incubated in a buffer containing 6.8 M urea, 62 mM Tris, 76 mM glycine-NaOH, pH 10, for 57 min, as previously described to remove FA/FB. 31 The PSI sample was then dialyzed overnight against 50 mM Tris-HCl pH 8.3. To remove FX, the sample was treated with 3 M urea, 5 mM K3FeCN6, 50 mM Tris-HCl, pH 8.0, for 4.5 h. 32 The PSI sample was dialyzed overnight against 50 mM TrisHCl, pH 8.3, and 5 mM 4,5-dihydroxy-1,3-benzene-disulfonic acid (disodium salt), and then again overnight against 2 changes of 50 mM Tris-HCl, pH 8.0, 0.03% β-DM. This sample was analyzed by ICP-AES analysis which showed a ratio of ~1 Fe/PSI monomer after urea treatment, confirming removal of the three Fe-S clusters. For EPR, the Fe-removed PSI sample was concentrated to 150 µM PSI monomer and with final conditions of 50 mM Tris-Cl, pH 8.3, 20% glycerol, 0.03% β-DM, and 10 mM sodium ascorbate. The chemically reduced Fe-removed PSI sample was prepared in 50 mM glycine-KOH pH 9.87, 20 % glycerol, 0.03% β-DM and 40 mM sodium hydrosulfite. EPR spectroscopy The samples were loaded into quartz tubes (inner diameter 0.5 mm / outer diameter 0.6 mm), dark-adapted for ~15 minutes at room temperature, and placed in the microwave cavity. The cavity was held in an Oxford flow cryostat, and temperature was controlled by the Oxford temperature control system. Dithionite-containing samples were cooled down to 205 – 245 K and illuminated with laser light (see below). All measurements were carried out at 100 K. EPR measurements were performed on a pulsed/continuous wave high-frequency, Dband (130GHz/4.6T) EPR spectrometer with single mode cylindrical cavity TE011.33-34 A fast pulse programming/acquisition system was developed by Dr. A. Astashkin, University of Arizona, on the basis of a 1 GHz arbitrary wave form generator PC card AWG1000 (Chase


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Scientific Co.). Pulsed TR-EPR spectra of the spin-correlated radical pairs (SCRP) were recorded by monitoring the electron spin echo (ESE) intensity from a two microwave pulse sequence, which followed a 10 ns laser pulse at a fixed delay after flash (DAF) time, as a function of magnetic field. Decay kinetics of the transient radical species were measured by recording the intensity of the ESE signal at a particular magnetic field value as a function of the DAF time. The duration of the π/2 microwave pulse was 40-60 ns, typical separation times between microwave pulses were 150-300 ns. Light excitation of the sample was achieved with an optical parametric oscillator (Opotek) pumped by a Nd: YAG laser (Quantel), the output of which was coupled to an optical fiber. The optical fiber allows delivery of up to 2 mJ per pulse to the sample. Excitation wavelength was 550 nm.

Results and discussion Specific samples were targeted to address the bidirectional nature of ET in PSI using time-resolved HF EPR (D-band, 130 GHz) spectroscopy: intact wild-type PS I and biochemically modified PSI RCs wherein the three terminal acceptor iron-sulfur centers, FX, FA and FB, have been removed to prevent secondary ET from phylloquinones A1 to Fx. To control for Fe-removal procedures by comparison to our previous work, one Fe-removed sample was treated with sodium hydrosulfite and then pre-illuminated at 205-245 K for 1 hr prior to data collection to reduce A1A. Each sample was prepared from fully deuterated Synechococcus leopoliensis; with deuteration aiding in the superior spectral resolution obtained with advanced HF EPR techniques by narrowing of the observed line widths. HF time-resolved EPR spectra for the three samples are shown in Figure 2A. Figure 2B shows the decay kinetics recorded at the quinone gx field position (signal at the lowest magnetic field), marked with an arrow in Figure 2A, as a function of electron spin echo intensity upon delay after laser flash (DAF-kinetics). All spectra were recorded at 100 K. A typical transient spin-correlated radical pair (SCRP) HF EPR signal for PSI is observed with the wild-type protein. (Figure 2Aa) This HF TR EPR signal has a well-known shape which originates from the P+A1A- pair due to photoinduced charge separation along A-branch. The rather complicated line shape of this SCRP signal depends upon the mutual orientations of the magnetic axis of the unpaired electrons in SCRP with respect to the inter-spin vector, thus different geometries of the SCRP are distinguishable enabling unique assignment of the charge separation state to either the A- or B-branches.20 For wild type PSI, the observation of P+A1Aradical pair is feasible because at low temperatures the electron transfer is blocked beyond the phylloquinone acceptor A1A. The SCRP from B-branch was never observed in wild type PS I. This was one of the strongest arguments for unidirectional electron transfer in Type I RCs through the A-branch.



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In previous work we blocked ET in PSI RCs from the cyanobacterium Synechococcus lividus by photo-chemical reduction of the FX electron acceptor.20 From this system, we were able to directly observe a SCRP from B-branch, P+A1B-. This HF EPR experiment provided unambiguous evidence of ET along Figure 2. HF TR-EPR data of PSI complexes from fully deuterated cyanobacterium Synechococcus leopoliensis. A. SCRP spectra recorded at 100 K, DAF-time 1 µs. Aa - intact the B-branch under wild type PS I. A-branch ET is blocked beyond quinone A1A. B-branch ET is irreversible and strongly reducing terminated at iron-sulfur complexes FA/B. Ab - Biochemically modified PSI RCs wherein the terminal acceptor iron-sulfur centers, FA/FB, and FX have been removed. This sample was conditions. However, treated with sodium hydrosulfite and pre-illumination at 205-245 K for 1 hr prior to data collection. This reduction procedure reduces the A1A quinone, and thus prevents ET along A questions remain branch. In the B-branch ET is blocked beyond A1B. Ac - Biochemically modified PSI RCs about the influence of wherein the terminal acceptor iron-sulfur centers, FA/FB, and FX have been removed; no prereduction conditions. In both A- and B-branches ET is blocked beyond quinones acceptors strong reducing A1A/B. Red – sum of two spectra: 44% of a (P+A1A-) + 56% of b (P+A1A-). B – Corresponding condition on the decay kinetics recorded at quinones' gX field positions, marked with arrows in A. Red curves – theoretical fits: Ba – 63 µs; Bb – 16 µs; Bc – 16 µs (50%) and 63 µs (50%). directionality of PSI ET. Moreover the pre-reduction procedure reduces the A1A acceptor, thereby preventing determination of the ratio of ET along A- and B-branches. Herein, we performed a similar experiment with PS I sample from fully deuterated Synechococcus leopoliensis wherein the terminal acceptor iron-sulfur centers FX, FA, and FB have been removed. One sample was treated with sodium hydrosulfite and then pre-illuminated at elevated temperatures to reduce A1A.12 A HF TR EPR spectrum of SCRP recorded at 100 K of this sample is shown in Figure 2Ab. The emission/absorption pattern of this spectrum is entirely different from that of wild type PS I (Figure 2Aa) and in very good agreement with B-branch SCRP spectra recorded earlier for PSI from Synechococcus lividus.20 Importantly, the decay of spin-polarization of two SCRP signals from A- and B-branches are different and can be fit with one exponent each: 63 µs for A-branch and 16 µs for B-branch as shown in Figure 2Ba and 2Bb respectively. These values are in good agreement with our previous data20 and indicate that the Fe-removal procedures do not impact the primary photochemistry of PS I.


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The SCRP spectrum of non-reduced Fe-removed PSI RCs is shown in Figure 2Ac (black). In this sample, if both branches are active for ET then the SCRP spectrum will be the sum of SCRP in A- and B-branches (P+A1A- + P+A1B-). Indeed, the polarization pattern of this signal is different from SCRP signal in the A-branch only (Figure 2Aa) and B-branch only (Figure 2Ab). The decay kinetics of this spin-correlated state (Figure 2Bc) can be fit as a biexponential decay with decay times of 63 µs and 16 µs, which clearly indicates that both Aand B-branches contribute to the spin-polarized pattern of the TR EPR signal in Figure 2Ac. The kinetic data on Figure 2Bc was fit with 1:1 ratio of pre-exponential factors. The low signal-tonoise ratio of the kinetic data does not allow for precise determination of the ET ratio along both branches. Therefore, the SCRP data was used to determine ET asymmetry. The SCRP of RC without iron-sulfur clusters was modelled as a sum of the SCRP spectra representative of A- and B-branch ET (Figure 2A). Spectra were normalized on the integrated intensity of the quinone’s low magnetic field signals (gX and gY components). The best fit to the experimental spectrum was obtained using a ratio of 44%:56% (A-branch:B-branch) as shown in Figure 2Ac (red). A 3% deviation from these values leads to a significant mismatch between the experimental and simulated spectra. These experiments provide direct evidence that the B-branch in PS I photosynthetic proteins is an active ET chain, even at low temperature (100 K). Surprisingly, the obtained ratio 44:56 (A:B) indicates that the main flow of the electrons proceeds through the B-branch and not the A-branch, as was postulated before. Note, utilization of both deuterated protein (line narrowing) and D-band EPR (high spectral resolution) made these conclusions possible. The SCRP spectra in Figures 2Aa and 2Ac would be nearly identical and unresolvable with the use of either protonated RCs or lower microwave frequency EPR spectroscopies.20, 23, 34-35 The bidirectional nature of ET in PS I and blocking of the A-branch ET beyond phylloquinone acceptor A1A at low temperature provides a straight-forward explanation of not only current data but also the suite of reported magnetic resonance and optical experimental data on native and mutant RCs at room and low temparatures.13-28, 31-32 Note, that if [4Fe-4S] cluster removal is the root cause of the observed in this work equal probability for ET along both branches, then the assertion of unidirectional ET in native RCs would require many strong assumptions to explain reported observations. One example of this are the well-known lightinduced EPR signals observed for dark-adapted PS I samples.23 PS I samples that have been dark-adapted at room temperature, frozen in the dark, and placed in a pre-cooled, dark EPR cavity exhibit no EPR signals. When these samples are illuminated in the EPR cavity at low temperature, strong stable EPR signals are observed and are identified as the radical species P700+, FA-, and FB –. These signals are difficult to quantify; rough estimates show that 50% or more RCs contribute to the signal intensity. Which pathway does the electron travel to reduce the terminal [4Fe-4S] clusters in wild-type RCs (dark-adapted)? As discussed above, electron transfer through the A-branch cannot proceed past the A1A acceptor at low temperature; and unidirectional ET model cannot explain this data. However, B-branch ET at low temperature can 7 ACS Paragon Plus Environment


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explain these results. Formation of FA- and FB- EPR signals is generated after ET through the Bbranch pathway of cofactors (not blocked beyond A1B at low temperature); which is consistent with our finding of 50/50 A/B branch ET at low temperature. This work also sheds light on the regulation of ET in photosynthetic RCs. Two possible mechanisms could determine the directionality of ET after photoexcitation of PS I. The first is a simple case of probability. There are two pathways; once a photon is absorbed the electron has a 50/50 chance of traveling down A- or B-branch. If this is the only factor, then at low temperature, the first flash 50% of RCs would have A-branch ET, 50% of RCs would have Bbranch ET. At low temperature, however, ET through the B-branch is non-reversible. Therefore, the “B-branch” RCs are trapped in the charge separated states P+FA- or P+FB-. No further turnovers can happen for this fraction (50%) of RCs. On the second flash, 25% of initial number of RCs would have A-branch ET, third flash, 12.5%, etc. A typical HF TR EPR spectrum is usually recorded after at least 1000 flashes. Therefore, with a 50/50 probability in wild-type PS I at low temperature after the first ~10 flashes or so all charge separation would irreversibly occur through the B-branch and a P+A1A- SCRP signal would not be observed. Note, that similar reasoning is valid for arbitrary ratio of probabilities, X/(1-X), only if X is not 0. Clearly, this scenario contradicts experimental data. A second possibility is related to protein’s inherent dynamical nature, with different conformational substates favoring A- vs B-branch ET. In this case, at low temperatures, the PSI RC is a nonergodic system, i.e. the average of ET events over time on one particular RC and the average over the statistical ensemble are not equivalent. This nonergodic formulation was successfully applied in the analysis of experimental results for both the temperature dependence of the ET rate and the nonexponential decay of the population of the photoexcited special pair in the R. sphaeroides purple bacterial RC.36 Relating this idea of nonergodicity to PS I, a certain fraction of RCs would exhibit ET only along the A-branch and another fraction would only have ET along the B-branch when frozen in solution. We hypothesize that the ratio of these “A-branch only” and “B-branch only” depends on conformational substates of the protein. These substates can be frozen at low temperature while coupled by protein dynamics at higher temperatures. Another plausible explanation is that “preprograming” occurs during the genesis or preparation of the RCs, is less favorable. We are planning to address this question in our forthcoming publication. In conclusion, advanced HF, TR EPR spectroscopy has been used to study transient SCRPs in PS I samples from fully deuterated Synechococcus leopoliensis: intact wild type PS I and biochemically modified PS I with the terminal Fe-S clusters removed. Results from these studies confirm the bidirectional nature of ET in PS I and furthermore reveal that the ratio of the electrons transferred along A- and B-branches is close to unity. Thus, this study provides important insight about features of Nature’s finely tuned solar energy conversion strategies in Type I photosynthetic RCs. Fundamental studies such as this can be applied to help develop


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strategies for and resolve proton-coupled electron transfer mechanisms in PSI-based hybrid systems for solar energy conversion.37-41 ACKNOWLEDGMENT

This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences, under contract number DE-AC02-06CH11357 at Argonne National Laboratory.



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TOC Figure. Using modified photosystem I reaction center protein wherein the acceptor ironsulfur centers, FA/FB, and FX, have been sequentially removed to prevent secondary electron transfer from phylloquinones (A1) to FX, it was demonstrated that at low temperature both A and B branches are active with the ratio close to unity.


Directionality of Electron Transfer in Type I Reaction Center Proteins

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