B-Branch Electron Transfer in the Photosynthetic Reaction Center of a

Mar 26, 2010 - W.L.: e-mail, [email protected]; fax, +49 208 306 3955; phone, +49 208 306 3614. K.M.: e-mail ... The directionality of light-...
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J. Phys. Chem. B 2010, 114, 14364–14372

B-Branch Electron Transfer in the Photosynthetic Reaction Center of a Rhodobacter sphaeroides Quadruple Mutant. Q- and W-Band Electron Paramagnetic Resonance Studies of Triplet and Radical-Pair Cofactor States† A. Marchanka,|,‡ A. Savitsky,‡,§ W. Lubitz,*,‡ K. Mo¨bius,*,‡,§ and M. van Gastel|,*,‡ Max-Planck-Institut fu¨r Bioanorganische Chemie, Stiftstrasse 34-36, D-45470 Mu¨lheim (Ruhr), Germany, Institut fu¨r Experimentalphysik, Freie UniVersita¨t Berlin, Arnimallee 14, D-14195 Berlin, Germany ReceiVed: January 13, 2010; ReVised Manuscript ReceiVed: March 11, 2010

The directionality of light-induced charge transfer in bacterial photosynthetic reaction centers (RCs) with respect to their A and B cofactor branches is still poorly understood on the electronic level. A prominent example is primary electron transfer in the RCs from the purple bacterium Rb. sphaeroides. Site-directed mutants with specific alterations of the cofactor binding sites with respect to the native system can deliver useful information toward a better understanding of the directionality enigma. Here we report on electron paramagnetic resonance (EPR) studies of the LDHW quadruple mutant, HL(M182)/GD(M203)/LH(M214)/ AW(M260), which contains crucial mutations in the electron-transfer pathway. The directionality of the charge separation process was studied under light- or dark-freezing conditions first directly by 95 GHz (W-band) high-field EPR spectroscopy examining the charge-separated radical pairs (P865•+QB•-) of the primary donor P865, a bacteriochlorophyll dimer, and the terminal acceptor, QB, a ubiquinone-10. Second, it was studied indirectly by 34 GHz (Q-band) EPR examining the triplet states of the primary donor (3P865) that occur as a byproduct of the photoreaction. At 10 K, the triplet state has been found to derive mainly from an intersystem crossing mechanism, indicating the absence of charge-separated radical-pair states with a lifetime longer than 10 ns. B-branch charge separation and formation of the triplet-state 3P865 via a radical-pair mechanism can be induced with low yield at 10 K by direct excitation of the bacteriopheophytins in the B-branch at 537 nm. At this wavelength, charge separation most probably proceeds via hole transfer from bacteriopheophytin to the primary donor. The triplet state of the primary donor is found to be quenched by the carotenoid cofactor present in the RC. The light-induced transient EPR signal of P•+QB•- is formed in a minor fraction of RCs ( 104 s) charge-separated-state P•+QB•-. The temperature dependence of the EPR signals from P•+QB•- points to two factors responsible for the forward electron transfer to the terminal acceptor QB and for the charge-recombination reaction. The first factor involves a significant protein conformational change to initiate P•+QB•- charge separation, presumably by moving the quinone from the distal to the proximal position relative to the iron. The second factor includes protein relaxation, which governs the chargerecombination process along the B-branch pathway of the LDHW mutant. Introduction To catalyze light-induced electron transfer in primary photosynthesis, reaction-center proteins of purple photosynthetic bacteria, such as Rhodobacter (Rb.) sphaeroides, carry two almost symmetrically arranged branches of protein-bound cofactors, A and B. Each branch contains an accessory bacteriochlorophyll (BChl), a bacteriopheophytin (BPheo), and a ubiquinone (Q); see Figure 1a.1-3 Photosynthetic charge separation in reaction centers (RCs) of the native system has been found to occur from a dimer of BChl molecules, called the primary donor, through a series of intermediate states that †

Part of the “Michael R. Wasielewski Festschrift”. * To whom correspondence should be addressed. W.L.: e-mail, lubitz@ mpi-muelheim.mpg.de; fax, +49 208 306 3955; phone, +49 208 306 3614. K.M.: e-mail, [email protected]; phone, +49 30 8385 2770. M.v.G.: e-mail, [email protected]; fax: +49 228 73 2551; phone, +49 228 73 2919. ‡ Max-Planck Institut fu¨r Bioanorganische Chemie. § Freie Universita¨t Berlin. | Present address: Institut fu¨r Physikalische and Theoretische Chemie, Rheinische Friedrich-Wilhelms Universita¨t Bonn, Wegeler Strasse 12, D-53115 Bonn, Germany.

involves the accessory BChl and the BPheo on the A-branch as well as both ubiquinones.2,4-9 This unidirectionality of electron transfer along the A-branch in wild-type RCs is still poorly understood on the electronic level. It has first been detected and investigated by optical spectroscopy at cryogenic temperatures, where the bands of the BPheo molecules in the A- and B-branches in the visible absorption spectrum become resolved. After light excitation, only one band bleaches, which means that only one BPheo is active in the charge separation.10 Linear-dichroism studies have shown that the active BPheo in charge separation is the one in the A-branch, abbreviated as HA,10-12 and electron transfer via the B-branch has been found to proceed with an upper limit of one percent.11,13-16 The molecular origin of the low yield of electron transfer via the B-branch in native bacterial RCs is still a matter of debate that has been refreshed owing to new achievements in site-specific mutations for photosynthetic bacteria like Rb. sphaeroides.17 They allow for detailed experiments to reveal the origins of the unidirectionality by breaking the 2-fold symmetry of the RC at several distinct places in the structure by introducing mutations in the amino-acid environment of the cofactors that

10.1021/jp1003424  2010 American Chemical Society Published on Web 03/26/2010

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Figure 1. Cofactor arrangement in the bacterial reaction centers from (a) Rb. sphaeroides 2.4.1 (PDB code: 4rcr), (b) Rb. sphaeroides double mutant LH(M214)/AW(M260), and (c) Rb. sphaeroides LDHW quadruple mutant LH(M214)/GD(M203)/LH(M214)/AW(M260).13 (d) Schematic cofactor arrangement for the quadruple mutant with nomenclature for each cofactor (for acronyms, see List of Abbreviations).

create differences in the hydrogen bonding network at corresponding cofactors in both branches.2 Another contribution might stem from differences in the local polarity of the specific cofactor environment by means of moving water molecules.18-20 A detailed understanding, on the molecular level, of the procedures nature employs to optimize the quantum yield of the light-induced electron-transfer steps in photosynthesis is also important for developing optimization strategies in synthetic chemistry aiming at novel donor-acceptor complexes with high quantum yield for efficient solar-energy conversion. Such an “artificial photosynthesis” approach is part of the worldwide efforts to develop carbon-neutral energy sources; for a recent review, see ref 21. Multiple spectroscopic studies have been performed in the past employing site-directed mutagenesis of Rb. sphaeroides to promote electron transfer through the B-branch.13,15,17,18,22-24 For example, a point mutation LH(M214) introduces a histidine near the BPheo a in the A-branch, whereby the BPheo a converts to BChl a (called also β). This mutated RC has been studied with optical techniques,25,26 and it has been found that the lifetime of the radicalpair-state P•+β•- shortens to 350 ps in comparison to 10 ns in RCs from wild-type Rb. sphaeroides.15,17,24,26,27 As was recently shown,28 the photosynthetic activity of the B-branch can be advantageously probed by electron paramagnetic resonance (EPR) spectroscopy of the excited triplet state (S ) 1) of the primary donor, 3P865. During photosynthetic charge separation in the native bacterial RC, formation of the triplet state is largely avoided.29 However, if the quinone cofactors are prereduced, for example, by adding sodium dithionite, or removed by site-directed mutagenesis, the photoexcited electron cannot travel further than to the bacteriopheophytin HA. The P•+HA•- radical pair formed upon excitation then decays to generate 3P865 with high yield.30 Since the radicalpair state is initially formed as a singlet state, it exclusively

decays into the MS ) 0 triplet sublevel of a triplet radical-pair state in about 10 ns at cryogenic temperatures.30,31 Hence, within this radical-pair (RP) mechanism of triplet formation, 3P865 is exclusively populated in its MS ) 0 state, and an EPR signal with a characteristic spin-polarization pattern AEEAAE (E ) emissive, A ) absorptive lines) is observed.32-35 Since it typically takes 10 ns to convert the singlet radical pair into a triplet radical pair, it is clear that EPR spectroscopy is essentially blind to radical-pair intermediates that recombine on a time scale shorter than 10 ns (for a recent overview of specifications of time resolution in modern EPR spectrometers, see ref 36). An ESE detected EPR spectrum of 3P865 recorded at 34 GHz and 10 K is shown in Figure 2a as an example. In a recent EPR study, the triplet states of the primary donor in the native RC of Rb. sphaeroides and that of the carotenoidless mutant R-26.1 as well as the two double mutants GD(M203)/ AW(M260) and LH(M214)/AW(M260) have been investigated.28 The GD(M203) mutation changes the H-bonding network near the accessory BChl a and makes the electron transport over the A-branch energetically less favorable. The mutation AW(M260) introduces a tryptophan in the binding pocket of QA, which blocks the access for the quinone at its native position.18,37 In RCs from Rb. sphaeroides 2.4.1, mutant R-26.1 and mutant GD(M203)/AW(M260), an AEEAAE spinpolarization pattern has been observed, which indicates the presence of a radical pair with a lifetime longer than 10 ns and a RP mechanism of triplet formation. In the mutant LH(M214)/ AW(M260) (see Figure 1b) a triplet state with the same zerofield-splitting (ZFS) parameters, but with a different polarization pattern (EEEAAA) and a strongly reduced EPR signal has been reported.28 This polarization pattern can only be explained by a spin flip of one of the unpaired electrons of excited singlet P* without a contribution of radical-pair intermediate states. This mechanism is known as intersystem crossing (ISC), and the

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Figure 2. (a) (Top) transient Q-band EPR spectrum and simulation of the RP formed triplet-state 3P865 in Rb. sphaeroides wild type; (middle) ESE detected EPR spectrum and its simulation of the ISC formed triplet-state 3P865 in the LH(M214)/AW(M260) double mutant;26 (bottom) ESE detected EPR spectrum and simulation of the RP formed triplet-state 3Car in Rb. sphaeroides wild type. Also depicted are the ZFS parameters D and E, the canonical orientations X, Y, and Z for 3P865 (P) and 3Car (C), and the polarization patterns (A ) absorptive, E ) emissive); for details see text. (b) ESE detected EPR spectra of the LDHW mutant at 10 K and excitation wavelengths of 865, 590, and 537 nm. (c) ESE detected EPR spectra of the LH(M214)/AW(M260) double mutant.

principal spin-dependent interactions responsible for the spin flip are spin-orbit (SO) coupling and spin-spin (SS) interaction.38 The observation of an ISC triplet state with EEEAAA polarization indicates that a radical-pair state is absent in both branches.28 Thus, even when charge-separated states at the A-branch with a lifetime longer than 10 ns are knocked out by the double mutations LH(M214) and AW(M260), it was found that charge separation on time scales longer than 10 ns at the B-branch still does not occur.28 The EPR spectrum of the LH(M214)/AW(M260) double mutant is included in Figure 2a. Thus, the polarization pattern of the triplet-state 3P865 gives unique information about the presence of precursor radical-pair states at the A and B branches. Moreover, in the RC of native Rb. sphaeroides, the triplet-state energy of 3P865 is taken over by the carotenoid (Car) cofactor to form triplet-state 3Car.39-41 The triplet-triplet energy transfer occurs at temperatures above 30 K and proceeds via the accessory BChl a in the B-branch.41-44 Since the sign of the ZFS parameter D of 3Car is opposite to that of 3P865, the spectra of 3Car and 3P865 have opposite polarization patterns, i.e., EAAEEA for the RP-formed tripletstate 3P865 (Figure 2a, bottom).28 For the ISC-formed triplet state, the polarization pattern of 3Car also depends on the relative orientations of the ZFS tensors of 3P865 and 3Car. Therefore, also the spin-polarization pattern of 3Car EPR spectra contains information about the presence or absence of radical-pair states at either the A- or the B-branch. High-field EPR experiments of the charge-separated radicalpair-state P•+QB•- in mutant RCs have been advantageously performed to understand the structure-dynamics-function relation of the RCs, concerning the spatial and electronic structures of the initial, final, and intermediate states. Standard 9.5 GHz (Xband) EPR spectroscopy of protein systems often faces problems of spectral resolution and sensitivity. In this situation, larger magnetic fields and microwave frequencies are needed to solve these problems and to select specific molecular orientations from the random distribution of the molecules in frozen solutions. For such disordered systems, millimeter and submillimeter highfield EPR methods, both in continuous wave (cw) and in pulse modes of operation, offer powerful tools for obtaining sufficient

spectral and orientational selectivity of the radicals and radical pairs and to provide the desired structural and electronic information. This holds particularly for radicals with largely varying interspin distances, for example, those generated transiently along the electron-transfer pathway between the redox cofactors arranged across the photosynthetic membrane.36,45-50 Subtle cofactor-protein interactions and/or conformational changes of specific protein segments, probably involved in fine-tuning the quantum yield of primary charge separation have been revealed by high-field EPR spectroscopy as well, and functionally important protein subdomains and cofactors have been characterized in terms of structure and dynamics in the hydrogen-bond network of the binding sites. Such pieces of information offer valuable additional information to that available from X-ray crystallography. In this work, a quadruple mutant24 of Rb. sphaeroides, called LDHW, is investigated. LDHW stands for the point mutations HL(M182)/GD(M203)/LH(M214)/AW(M260) (see Figure 1c). The directionality of the charge-separation process was studied under light- or dark-freezing conditions by two different EPR experiments: (i) directly by 95 GHz (W-band) high-field EPR spectroscopy examining the charge-separated radical pairs, P•+QB•-, of the primary donor P865, a BChl a dimer, and terminal acceptor, QB, a ubiquinone-10, and (ii) indirectly by 34 GHz (Q-band) EPR examining the triplet states of the primary donor, 3 P865, that occur as byproduct of the photoreaction. In this case, the spin-polarization pattern of the triplet EPR spectrum contains important information about the precursor radical-pair states. The cofactor arrangement and the mutations are shown in Figure 1c. For comparison, the cofactors in the Rb. sphaeroides wild type (Figure 1a) and in the LH(M214)/AW(M260) double mutant (Figure 1b) are also shown. Three of the four mutations, GD(M203), LH(M214), and AW(M260), are identical to those of the aforementioned mutants. The fourth mutation HL(M182) is the replacement of histidine by leucine by which the accessory bacteriochlorophyll BB is changed to a bacteriopheophytin,ΦB. It has been observed that the presence of ΦB leads to an increased B-branch electron transfer,22,23,51 and a yield of up to 40% at room temperature has been reported.24 Moreover, both

Photosynthetic Reaction Center of Rb. sphaeroides bacteriopheophytins in the B-branch have been found to participate in the charge-separation process.24 The recombination times of the radical pairs P•+HB•- and P•+ΦB•- at 10 K were found to be similar and below 1 ns.24 This time constant is larger than that of the Rb. sphaeroides single mutant HL(M182)22 for which a recombination time constant of 200 ps at room temperature has been reported. The aim of the present study is to detect long-lived charge separated states at the B-branch of the LDHW mutant using the triplet state, the doublet state and the radical-pair state of the primary cofactors. We characterize these paramagnetic states in terms of electronic structure and formation/decay kinetics and probe the efficiency of B-branch electron transfer in the LDHW mutant, by EPR spectroscopy at 34 and 95 GHz. Materials and Methods Sample Preparation. (i) Samples for Triplet-State Q-Band EPR Measurements. The Rb. sphaeroides mutant LDHW was expressed and purified according to de Boer et al.24 In their work, the mutagenesis system originally developed by Paddock et al.52 has been used. EPR measurements on the triplet states were performed on samples treated with the herbicide terbutryn as an inhibitor that binds in the QB site. It was observed that the addition of tetbutryn yields the same EPR spectra of the triplet state as those for samples without terbutryn; however, the terbutryn prevents the buildup of photoaccumulated P•+QB•with QB in the distal position.24 The samples have been frozen in liquid nitrogen in the dark. The optical density of the samples was 50 at 865 nm and the purity of the samples was verified by UV/vis measurements at room temperature. (iı´) Samples for Radical-Pair-State W-Band EPR Measurements. The samples for the W-band EPR measurements on the radical-pair-state P•+QB•- did not contain terbutryn. Also, the samples were not reconstituted with additional quinones and contained the native Fe instead of the usually used Zn. The Fe was retained not to disturb the RC other than with the four mutations. It does give rise to fast spin relaxation, which limits the time to detect the signal by pulsed EPR spectroscopy. Consequently, the W-band EPR measurements required the detection sensitivity and time resolution to be optimized (see ref 36), for detecting the transient radical pairs. Pulse EPR Methods. (i) 34 GHz Measurements. Electron spin echo (ESE) detected EPR spectra have been recorded on a Bruker Q-band Elexsys E580 FT pulse EPR spectrometer with pulsed laser excitation as described previously.28,53 Excitation at 537 nm has been performed with 9 mJ/pulse, at 590 nm with 7.5 mJ/pulse, and at 865 nm with 3.0 mJ/pulse. The EPR experiments have been performed in the temperature range 10-150 K. The two-pulse EPR and delay-after-flash (DAF)EPR schemes consist of a laser pulse followed by two microwave pulses and then detection by a Hahn echo.54 In the ESE detected EPR experiments the magnetic field is swept. In DAF-EPR, the magnetic field is fixed and the delay time between the laser pulse and the first microwave pulse is stepwise incremented. Chosen pulse lengths and delay times have been given elsewhere.28 The accumulation time was typically 0.5-2 h for an EPR spectrum, depending on the temperature at which the measurements have been performed. Simulations of the Q-band EPR spectra of the triplet state were performed with a self-written program28 based on the formalism described in reference.55 (ii) 95 GHz Measurements. High-field EPR measurements have been performed on a laboratory-built W-band spectrometer that had been optimized for a variety of cw and pulse

J. Phys. Chem. B, Vol. 114, No. 45, 2010 14367 experiments, as described previously.36,48 The spectrometer was equipped with a TE011 optical transmission cavity with unloaded quality factor QU ) 5000. For optical sample irradiation the light was guided to the center of the cavity through a quartz fiber of 0.8 mm diameter. The electron transfer was initiated by singlet excitation of the primary donor at 532 nm using a Nd:YAG laser (5 ns pulse length, 10 Hz repetition rate, 0.5 mJ/pulse on the sample surface) or at 690 nm using a cw diode laser (25 mW output, 10 mW on the sample surface). The pulse EPR measurements were performed using the microwave pulse sequence for primary echo generation: (tp)x,-x-τ-(2tp)-τ-echo. The pulse length of the π/2 mw pulses and the pulse separation time τ were generally set to 30 and 150 ns, respectively. The time-resolved W-band EPR measurements of short-lived paramagnetic transients were performed using the direct-detection technique with a time resolution of 10 ns. The accumulation time for an EPR spectrum was typically 5 min depending on the sample temperature. All experimental spectra analysis and simulation procedures were performed on the basis of the EasySpin toolbox for the Matlab program package.56,57 Results and Discussion Q-Band Triplet-State EPR Spectra at 10 K with Light Excitation at 865, 590, and 537 nm. The Q-band ESE detected EPR spectra of a dark-frozen sample of RCs from the quadruple mutant LDHW at 10 K, excited at 537, 590, and 865 nm, are shown in Figure 2b. The EPR spectra of the triplet state are characterized by polarization patterns EAEEAAEA with light excitation at 537 and 590 nm, and EEEEAAAA at 865 nm. A polarization pattern with more than six canonical orientations indicates the presence of several paramagnetic species. The D and E values extracted from the spectra agree with those of 3 P865 and 3Car of Rb. sphaeroides wild type.26 Since the time constants of formation and decay of 3P865 and 3Car are different, EPR spectra have been recorded at different time intervals between light excitation and detection to assign the signals to 3 P865 and 3Car, respectively. The assignment is included in Figure 2. The EPR spectrum displays significantly smaller amplitudes at the absorptive transitions of the canonical orientations XI, YI, ZI as compared to the emissive transitions. This feature appears to be characteristic for Q-band EPR on 3P865 at low temperatures and can be explained by the ESEEM effect, which may introduce a modulation of the ESE amplitude in pulsed EPR spectroscopy owing to the presence of nearby nuclear spins, in particular those of 14N (I ) 1).28 A comparison of the EPR spectra of RCs from the LDHW quadruple mutant with those from the Rb. sphaeroides wild type and the double mutant LH(M214)/AW(M260)28 is given in Figure 2a-c. With light excitation at 537 nm, the EPR spectrum of the LDHW mutant is similar to that of the wild type at 10 K.28,32,35 In the LDHW mutant the amount of RP-formed triplet state is estimated to be equal to the amount of ISC-formed triplet state, whereas in the LH(M214)/AW(M260) mutant only a minor contribution of a RP-formed triplet state was observed at 537 nm excitation. At 590 nm, the amplitudes of all EPR signals decrease, those at the YI and YII canonical orientations decrease even more than those at the XI and XII orientations. Since the RP mechanism induces strong signals at YI and YII (see Figure 2a), their absence indicates that less radical pairs have been formed at this excitation wavelength. In the LH(M214)/ AW(M260) mutant, excitation at 590 nm yields a triplet state, which is almost completely derived from the ISC mechanism. At 865 nm excitation the EPR spectrum of the LDHW mutant is similar to that of the LH(M214)/AW(M260) mutant and also

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Figure 3. Q-band ESE detected EPR spectra of the LDHW mutant at (a) 30 K and (b) 50 K, recorded at excitation wavelengths of 865, 590, and 537 nm.

almost exclusively displays ISC character. This indicates that virtually no radical-pair states with a lifetime longer than 10 ns are present in both branches to generate triplet states. Apparently, direct excitation of P865 at 865 nm in the LDHW mutant does not induce radical-pair precursor states sufficiently longlived for forming 3P865, whereas excitation at 590 or 537 nm does. It is interesting that in the LDHW mutant, the carotenoid is able to take over the triplet excitation already at 10 K, whereas in the native and different double mutant systems of Rb. sphaeroides an activation barrier for this triplet-energy transfer of 30 K had been found.42,43 The amplitude of the 3Car signal also depends on the excitation wavelength. Since 3Car has a larger D parameter than 3P865, the presence of 3Car is easily recognized at the low-field and high-field edges of the EPR spectrum. As seen in Figure 2, a significantly stronger 3Car triplet signal is observed with excitation at 537 and 590 nm than at 865 nm. This observation may be related to the fact that the RP mechanism, operative at 537 and 590 nm, induces maximum spin polarization because only the MS ) 0 sublevel is populated and the other sublevels are not populated at all, whereas in the ISC mechanism the spin population is distributed over all triplet sublevels, leading to a smaller spin polarization of the EPR spectra. Q-Band Triplet-State EPR Spectra at Elevated Temperatures. EPR spectra of the triplet state of the LDHW mutant at 30 K, excited at 537, 590, and 865 nm, are shown in Figure 3a, those at 50 K in Figure 3b. As compared to the spectra at 10 K, the signals at 30 K have become less intense. The EPR spectrum recorded with excitation at 865 nm now has increased amplitudes at YI and YII. Thus, with excitation at 865 nm, the EPR spectra of the triplet state obtain more RP character at elevated temperatures. At 50 K, the EPR signals become very weak. The spectra are largely similar to those recorded at 30 K. The contribution of 3Car to the signal has increased relative to that of 3P865, which indicates that the carotenoid takes over the triplet excitation faster at elevated temperatures. The triplet-excitation transfer to 3Car observed for the LDHW mutant is slower than for the LH(M214)/AW(M260) mutant, for which a complete quenching of the 3P865 signal has been observed at 50 K.28 Q-band EPR measurements have also been performed at 100 and 150 K (see Supporting Information). At these elevated temperatures the triplet signals become even weaker, and only noisy spectra could be recorded. At 100 K, the shape of the

Marchanka et al. spectra becomes independent of the excitation wavelength and attains more RP character; a residual amount of 3P865 could still be detected in the EPR spectra, whereas at 150 K only the signals from 3Car are present. Though the amount of information extracted from these spectra is rather limited given their small signal-to-noise ratio, apparently long-lived radical-pair states are present at elevated temperatures, independent of the excitation wavelength. Kinetics of the Triplet States. The decay kinetics of the EPR signals from 3P865 and 3Car has been studied at different temperatures. The 3P865 and 3Car signals could be distinguished by DAF-EPR experiments (see above) since the time constants for growth and decay of the 3P865 and 3Car signals are different. The decay constants of the 3P865 signal of the LDHW mutant are included in Table 1 and are virtually identical to those of Rb. sphaeroides wild type and double mutants.28 However, at 10 K the carotenoid shows decreased decay constants, especially at the Y canonical orientation (2.5-3.0 µs), which is smaller by a factor of 6 as compared to the decay constant (15 µs) in the RCs from the wild-type and the double mutants LH(M214)/ AW(M260) and GD(M203)/AW(M260).26 The decay constants of 3Car at the Z and X canonical orientations (9.6 and 27 µs) are also smaller than those for Rb. sphaeroides wild type (15 and 45 µs). Since the only mutation near the carotenoid involves the exchange of BB to ΦB, it is tempting to speculate that the faster decay of the 3Car signals may be caused by distortion of the binding pocket and increased dynamics of the carotenoid cofactor. W-Band EPR Characterization of the Radical-Pair-State P•+QB•- Formation along the B-Branch. It is important to first mention again that the RC samples for the radical-pair measurements by W-band EPR did not contain terbutryn which would inhibit QB binding and, hence, formation of P•+QB•-. In the previous investigation of the LDHW mutant24 it has been shown by UV/vis spectroscopy that upon light excitation at room temperature a mixture of the radical-pair-states P•+HB•- and P•+ΦB•- is formed with a yield of about 40%. At this temperature, the forward electron transfer directly to the QB quinone acceptor, leading to P•+QB•- formation, occurs to a minor extent of about 1%.24 Although the P•+QB•- state is formed with such a low yield, prolonged illumination of the LDHW sample is expected to result in an appreciable population of a photoaccumulated long-lived P•+QB•- state. A typical lifetime of P•+QB•of several seconds in RC mutants void of QA had been reported previously.58 The samples were either frozen in the dark after 10 min of dark adaptation at room temperature (dark-adapted RCs) or frozen under continuous illumination starting from room temperature (light-adapted RCs). As seen in Figure 4a, the darkadapted sample reveals no background signal in the W-band field-swept ESE detected EPR spectrum. Under illumination with a cw laser at 690 nm a weak EPR signal appears that consists of only one slightly asymmetric line positioned around g ∼ 2.0026. This line is attributed to the primary donor radical cation of P865, P•+. This signal decays within several seconds when actinic illumination was terminated. In contrast to the darkadapted sample, the sample frozen under illumination shows an intense EPR signal (Figure 4b). Simulations of the EPR line shape in the high-field region yield the principal g-tensor components 2.00322, 2.00234, and 2.00194, which are in good agreement with previously reported gxx, gyy, and gzz components for P•+.49,59-61 An additional signal is present in the low-field region of the spectrum. It is attributed to the radical anion of the B-branch quinone electron acceptor, QB•-, with the gxx and

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TABLE 1: Decay Constants (µs) for the 3Car Signal in the EPR Spectra of the LDHW Mutant, Measured at the Canonical Orientations (X, Y, Z) and at Variable Temperaturesa decay constants 3

3

3

P865 (LDHW)

3

Car (wt)

Car (LDHW)

T (K)

X

Y

Z

X

Y

Z

X

Y

Z

10 30 50 100

80

80

250

33

33

63

47 25.2 14.7 11.8

15 9.2 5.6 3.3

15 9.2 5.6 3.3

27.0 9.0 8.1 8.7

2.5 2.5 3.0 3.0

9.6 7.6 6.0 7.8

a Also included are the decay constants of 3P865 and those for 3Car in the native (wt) RC. The accuracy is about (4 µs for 3P865, (0.5 µs for Car (wt), and (0.9 µs for 3Car (LDHW).

Figure 4. Field-swept echo detected W-band EPR spectra recorded at 90 K for the LDHW mutant RCs: (a) frozen in the dark; (b) under continuous illumination. Light-off and light-on spectra are shown by green and blue solid lines, respectively. The samples were illuminated with a cw laser (690 nm). The inset in (b) shows an extended QB•- spectral region. The simulation with [gxx; gyy] ) [2.00623; 2.00521] is given by the dotted line. For details, see text.

gyy values of 2.00623 and 2.00521, as obtained from spectral simulations; see Figure 4b (insert). Since the nonheme iron was not replaced in the LDHW sample, the EPR signal of QB•- is only visible in the small fraction of RCs that lost the iron upon preparation and, therefore, its EPR signal is not broadened by the magnetic interaction of the semiquinone with high-spin Fe2+. The integrated intensity of the QB•- signal is about 3% of the P•+ signal, which is a typical ratio for Fe2+ containing RC samples. After the continuous illumination had been switched off, about 96% ((1%) of the EPR signal of the dark-adapted sample was found to be stable at 90 K; i.e., the photoaccumulated radical fraction did not decay for a long time (τ > 104 s). Thus, the dark-adapted LDHW mutant RCs at 90 K are predominantly trapped in a conformation that is “inactive” for cyclic electron transfer, i.e., in which the transient P•+QB•- state is not formed. This observation is in agreement with earlier results of de Boer et al.24 Only a small portion of the EPR signal of about 4% was transient in character and did decay with a time constant of about 5 s. A comparison of the absolute EPR intensities for dark-adapted and light-frozen samples (see Figure 4) shows that in the dark-adapted sample the transient P•+QB•state is formed only in about 1% of the RCs relative to the formation of transient P•+QB•- in the light-frozen sample. This minor RC fraction in dark-adapted samples is trapped in an “active” conformation able to produce light-induced the transient P•+QB•- state (τ ∼ 5 s) at 90 K. In contrast, in light-adapted RCs the largest fraction of RC’s is trapped in an “active” conformation. To estimate the absolute fraction of RCs which are able to form the P•+QB•- radical pair, transient W-band EPR intensities of the triplet states after the laser flash illumination at 532 nm were compared. The amplitude of the transient responses of the light-frozen sample was about 3-fold smaller compared to the dark-adapted one. This indicates that in the

light-adapted sample about 70% of the RCs are forming the P•+QB•- state upon illumination. This situation for the light-frozen LDHW sample is similar to that recently reported for a series of Rb. sphaeroides mutant RCs carrying the AW(M260) mutation motif.58 This mutation prevents A-branch electron transfer because such mutants lack QA. These authors observed that the stable (τ > 107 s) P•+QB•radical-pair state is formed in about 95% of the mutant RCs, which were frozen to 80 K under illumination (light-adapted RCs). However, different from our findings for the LDHW mutant, their mutant samples when frozen in the dark (darkadapted RCs) reveal cyclic activity toward formation of the transient (τ ) 6 s) P•+QB•- radical pairs in as much as 30% of RCs. The authors concluded that in the dark-frozen sample (ground-state protein conformation) about 70% of the QB molecules in the RCs are present in the “distal” position (with respect to the iron) where they are not accessible for electron transfer, i.e., stay “inactive”, whereas the remaining 30% fraction of the RCs are in the “active” conformation in which QB occupies the “proximal” site with respect to the iron. On the other hand, it was observed58 that in the sample frozen under illumination (charge-separated-state conformation) about 95% of the RCs become trapped with QB located in the “proximal” site. The large difference of P•+QB•- radical-pair lifetimes in both samples, dark-adapted and light-adapted, was attributed to the protein relaxation, in response to light-induced charge separation, and internal water molecules that stabilize P•+QB•in the light-adapted sample (“relaxed” P•+QB•- state).58 In a subsequent paper it was reported that part of such protein relaxation involves the formation of an H-bond between the OH group of Ser-L223 and the anionic semiquinone QB•-.62 Also our results on the LDHW mutant RCs can be explained on the basis of this proposed model.58 Specifically, we have to

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Figure 5. Field-swept echo detected W-band EPR spectra recorded for the LDHW mutant RCs: (a) frozen to 90 K under continuous illumination; (b) warmed to 180 K in the dark. Light-off and light-on spectra are shown by green and blue solid lines, respectively. The samples were illuminated with a cw laser (690 nm).

assume that in the dark-adapted sample QB occupies almost exclusively the “inactive” distal site, and that in the light-adapted sample QB can be trapped in the “active” proximal site only in about 70% of RCs under our experimental conditions. The long lifetime of the trapped P•+QB•- state shows that the LDHW RCs are predominantly frozen in the “relaxed” P•+QB•- state in which the protein relaxation lowers the energy of the charge-separated state to such an extent that the charge recombination does not occur any more at 90 K. The protein relaxation may be caused by a large variety of small perturbations including H-bond formation, protein rearrangements, proton or water molecule movement, charge relaxation, etc. Thus, the transition between relaxed (light-adapted) and unrelaxed (dark-adapted) states can be expected to be temperature dependent, even below the glass-transition temperature of the protein. This temperature dependence was indeed demonstrated by studying the charge recombination between P•+QA•- in wild-type RCs from Rb. sphaeroides.63,64 In contrast, the largescale protein conformation changes, for example, QB movement between proximal and distal positions, takes place at temperatures above the glass-transition region, i.e., above 200 K. We should point out that protein relaxation upon excitation and charge separation is now generally considered necessary for understanding electron transfer in photosynthetic RCs.64-68 To separate effects of protein relaxation from possible influences of different occupancy of the proximal and distal QB positions, additional temperature dependent W-band EPR experiments were performed. Both frozen samples, dark-adapted and light-adapted, were warmed up in the dark from 90 to 180 K, thus allowing for partial protein relaxation, but still prohibiting conformational changes of the QB site. The samples were characterized by field-swept ESE detected EPR before, during and after cw illumination at 690 nm. Comparison of the transient EPR intensities at 90 and 180 K, taking into account the decreased Boltzmann population difference at higher temperatures, allows us to quantify the activity for cyclic electron transfer. For the dark-adapted sample the activity for transient P•+QB•- formation was still not larger than about 1.5 ( 0.5% at 180 K (it was 1% at 90 K). Thus, the dark-adapted sample shows only negligibly increased activity of transient P•+QB•formation at higher temperature, telling us that in this situation of a “frozen-in” ground-state RC conformation protein relaxation is not yet a relevant process for light-induced electron transfer at 180 K. The situation for the light-adapted sample, i.e., frozen under illumination, is different: the fraction of RCs able to form the transient P•+QB•- state increases from 4% (90 K) to 21% (180 K). This result was obtained from a comparison of the EPR intensities in the P•+ and QB•- spectral regions before, under

and after the actinic illumination; see Figure 5. Thus, in lightadapted LDHW samples the “inactive” fraction of RCs with trapped long-lived P•+QB•- radical pairs diminishes in favor of “active” RCs able to form transient P•+QB•- pairs, meaning that protein relaxation plays an important role for light-induced electron transfer. Summary and Conclusions In this contribution, Q-band and W-band EPR studies on triplet and radical-pair cofactor states in RCs from the Rb. sphaeroides LDHW mutant have been performed for a better understanding of primary B-branch charge separation and to investigate HOMO- and LUMO-based electron transfer, as summarized in Figure 6. In this mutant, the A-branch comprises two BChl a cofactors, the B-branch two BPheo a cofactors. This configuration of cofactors has been used to selectively investigate the presence of charge-separated intermediate states with a lifetime longer than 10 ns at either of the two branches. At 10 K and 865 nm light irradiation, the excited singlet-state P865* of the primary donor does not give rise to charge-separated intermediate states with a lifetime longer than 10 ns at both branches, and an excited triplet state of the primary donor 3P865 almost exclusively derived from the ISC mechanism is observed (Figure 6a). The same observation is made for the double mutant LH(M214)/AW(M260),28 indicating that the B-branch does not take over photosynthetic activity at these temperatures even when charge-separated states at the A-branch were eliminated owing to site-directed mutations. Selective excitation of the bacteriopheophytins HB and ΦB in the B-branch at 537 nm does lead to the presence of RPbased triplet EPR signals. The singlet-excited states HB* and ΦB* have one unpaired electron in the HOMO and one in the LUMO, or alternatively, one hole in these orbitals. In this case, it is possible that an electron is donated by P to fill the hole in the HOMO. This type of electron transfer is thus HOMO based and is also called hole transfer, as opposed to the direct excitation of P, where the electron transfer from P to Φ is LUMO based. The radical-pair states P•+HB•- and P•+H(Φ)B•are in this case formed by hole transfer into the HOMO orbital from HB and ΦB to P865 (Figure 6b). Selective excitation of the bacteriochlorophylls BA and β in the A-branch at 590 nm also leads to radical pairs, but to a lesser extent than upon excitation of the bacteriopheophytins. At elevated temperatures the wavelength dependence observed in the EPR spectra diminishes, which points to a higher yield of radical pairs with a lifetime larger than 10 ns, formed

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Figure 6. Proposed mechanism of the energy and electron transfer and charge separation in the Rb. sphaeroides quadruple mutant LDHW at (a) 865 nm excitation and (b) at 537 nm excitation. The blue arrow depicts fast energy transfer from bacteriopheophytins to the primary donor; the purple arrow indicates the hole transfer mechanism involved in the RP formation. The orange arrow concerns the ISC mechanism of triplet formation, and the green arrows indicate the pathways for the RP formed triplet states. For details, see text.

directly from singlet-excited P865*. Simultaneously, the carotenoid becomes more efficient in taking over the triplet excitation, which leads to an overall decreased amplitude of the EPR signal of 3P865 and an increased signal of 3Car. Though it should be stressed again that EPR spectroscopy of 3P865 is not able to detect radical pairs with recombination times shorter than 10 ns, it is the longer-lived radical pairs that are important for forward electron transfer. The observations that HOMO based electron/hole transfer does lead to radical pairs at 10 K, and that both RP and ISC mechanisms of triplet formation are operative at elevated temperatures, suggest that HOMO and LUMO based charge separation likely plays a role in parallel in native RCs at ambient temperatures. Apparently the cofactor arrangement in the bacterial RC has been optimized during evolution to such an extent that the complete excitation wavelength region covered by P865, BChl a, and BPheo a leads to photosynthetic charge separation, be it either by LUMO or by HOMO based mechanisms, and exclusively in the A-branch. Additional information about B-branch electron transfer in the LDHW mutant has been obtained by W-band EPR spectroscopy of the light-induced radical-pair state P•+QB•-. The RC samples were cooled to 90 K either in the dark or under continuous illumination. In the mutant RC frozen in the dark, the light-induced transient EPR signal of P•+QB•- is formed in a minor fraction of RCs ( 104 s) charge-separated state P•+QB•- The temperature behavior of the EPR signals from P•+QB•- points to two factors responsible for the forward electron transfer to the terminal acceptor QB and the charge-recombination reaction. The first factor involves a significant protein conformational change to initiate P•+QB•charge separation, presumably by moving the quinone from the distal to the proximal position relative to the iron. The second factor includes a protein relaxation process affecting the chargerecombination process along the B-branch pathway of the LDHW mutant. The combined study of triplet and radical-pair states by EPR spectroscopy has revealed several important factors that govern electron transfer in reaction-center proteins. First, the photosynthetic cofactors in the A-branch have been optimized for charge separation as compared to those in the B-branch, with respect to both the orbital overlap and the energies of the respective HOMO and LUMO orbitals. Second, protein dynamics are important, especially with respect to the binding pocket of the final electron acceptor, QB, which can be present in at least two conformations. Third, upon charge separation, the changed electronic configuration causes local changes in the binding pockets, denoted as protein relaxation, which may

inhibit the back reactions. It is still unclear which mutations are necessary to increase the electron-transfer matrix elements of the cofactors in the B-branch. One possibility to address this question would be to study the HOMO and LUMO orbitals of the isolated cofactors by ENDOR spectroscopy53 and to selectively study the cofactors in single crystals by excitation at the appropriate wavelengths. Acknowledgment. We are grateful to Dr. P. Gast (Leiden University) who provided the sample of the LDHW mutant. This project was supported by a DFG-NWO international collaborative research initiative, specifically by the DFG grants GA 1100/1-2 and MO 132/19-2. List of Abbreviations BB BChl a BPheo DAF EPR ESE ΦB H HB ISC LDHW P 3 P865 P•+QB•Q Rb. RP SO SS ZFS

bacteriochlorophyll in the B-branch bacteriochlorophyll a bacteriopheophytin delay after flash electron paramagnetic resonance electron spin echo “new” bacteriopheophytin in the B-branch bacteriopheophytin bacteriopheophytin in the B-branch intersystem crossing HL(M182)/GD(M203)/LH(M214)/AW(M260) primary donor triplet primary donor in Rhodobacter sphaeroides Radical pair of primary donor and final quinone acceptor quinone Rhodobacter radical pair spin-orbit spin-spin zero field splitting

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