Orientation Resolving Dipolar High-Field EPR Spectroscopy on

Article pubs.acs.org/JPCB

Orientation Resolving Dipolar High-Field EPR Spectroscopy on Disordered Solids: II. Structure of Spin-Correlated Radical Pairs in Photosystem I A. Savitsky,*,† J. Niklas,† J. H. Golbeck,§ K. Möbius,†,‡ and W. Lubitz† †

Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36, D-45470 Mülheim an der Ruhr, Germany Department of Physics, Free University Berlin, Arnimallee 14, D-14195 Berlin, Germany § Department of Biochemistry and Molecular Biology, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡

S Supporting Information *

ABSTRACT: The distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on electron transfer proteins, we report the results of orientation resolving dipolar high-field •− EPR spectroscopy on the charge-separated state P•+ 700A1 (P700, primary electron donor; A1, phylloquinone electron acceptor) in Photosystem I (PS I). Pulsed high-field EPR spectroscopy at W-band (95 GHz, 3.4 T) with extensions to PELDOR (pulsed electron−electron double resonance) and RIDME (relaxationinduced dipolar modulation enhancement) was utilized to •− obtain the parameters describing the three-dimensional structure of the laser-flash-induced transient radical pair P•+ 700A1 in a frozen solution of deuterated PS I from the cyanobacterium Synechocystis sp. PCC 6803, which is performing oxygenic •− photosynthesis. The measured distances and relative orientations of the weakly coupled radical ions in the radical pair P•+ 700A1 are compared with previously reported geometries and with those of the precursor cofactors P700 and A1 known from X-ray crystallography. Cyclic electron transfer was found to proceed exclusively via the A-branch of the cofactor chain of PS I at cryogenic temperature. The position and orientation of the reduced phylloquinone coincide with those of the precursor, revealing that no substantial orientational changes of the phylloquinone molecule upon charge separation occur. Several distinct orientations of the P•+ 700 g-tensor axes with respect to the molecular frame of the primary donor were found experimentally, which we explain by several conformational substates of the P•+ 700 radical structure having slightly different electron spin density distributions.

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INTRODUCTION

interaction parameters and the geometry of the system can be obtained. Three-dimensional geometrical information about the radical pair is of particular importance. It allows one to extract structural information about the transient charge-separated states in photosynthetic reaction centers (RCs) for which only rarely13 detailed X-ray data are available. In principle, EPR methods enable one to recognize and characterize the small structural changes that might occur in RCs as a result of chargeseparation and charge-recombination processes. Although the time-resolved EPR signals of the SCRP state are very sensitive to the relative orientation of the radicals in the pair, the number of parameters on which the SCRP EPR spectrum depends is

Light-induced transient radical pairs in photosynthetic reaction centers of plants and bacteria have been the focus of numerous spectroscopic investigations over the past decades.1−3 In particular, the charge-separated radical pairs P•+Q•− of the primary electron donor and either one of the quinone acceptors have been characterized by a variety of time-resolved EPR methods3−6 in the bacterial reaction center (bRC),7−9 as well as in plant photosystem I (PS I)10,11 and photosystem II (PS II).12 After pulsed laser excitation of the primary donor, P•+Q•− appears in the spin-correlated coupled radical-pair (SCRP) state, which is characterized by a weak electron spin−spin dipolar coupling in a fixed geometry of the radicals in the pair and an initial singlet state of the system.9 The EPR responses of the SCRP state display a number of interesting and useful spectroscopic features: spin polarization, quantum beats, transient nutations, as well as echo-envelope modulation and out-of-phase echo effects.2 From their analysis, the magnetic © XXXX American Chemical Society

Special Issue: Rienk van Grondelle Festschrift Received: February 13, 2013 Revised: April 9, 2013

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dx.doi.org/10.1021/jp401573z | J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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large, and for unique solutions of the spectral simulations additional independent information is needed. Hence, it is often difficult to judge the accuracy of the extracted pair geometry. Therefore, an additional EPR methodology ought to be employed, which reveals information on the orientation directly, and this with high accuracy. This methodology is pulsed electron−electron double resonance (PELDOR or DEER) dipolar spectroscopy.14,15 It uses two microwave frequencies, but at high magnetic fields we prefer to perform it in conjunction with the one-frequency RIDME (relaxationinduced dipolar modulation enhancement) dipolar spectroscopy technique16,17 which delivers already preliminary orientation information. Such information is very useful to choose proper instrument settings for the PELDOR experiment.18,19 Recently, we have demonstrated that by combined pulsed high-field EPR, RIDME, and PELDOR methods in conjunction with elaborate data-analysis procedures the distance and orientation of transient radical pairs in electron-transfer proteins can be determined with high accuracy.18 Taking the •− donor−acceptor radical pairs P•+ 865QA in frozen-solution bRCs from the nonoxygenic photosynthetic purple bacterium Rhodobacter (Rb.) sphaeroides as an example, it has been demonstrated that such pulsed high-field dipolar EPR and PELDOR experiments have reached a level of sophistication wherein details of the three-dimensional molecular structure of transient short-lived reaction intermediates can be elucidated to a precision comparable to that of high-resolution protein X-ray crystallography. In the work presented here, W-band (95 GHz) high-field (3.4 T) dipolar EPR techniques such as PELDOR and RIDME were applied to transient radical pairs of the charge-separated •+ primary electron donors P700 and acceptors A1•− in the photosynthetic reaction center PS I from the oxygenic cyanobacterium Synechocystis sp. PCC 6803. PS I of cyanobacteria, algae, and higher plants is a transmembrane protein complex that mediates light-induced electron transfer across the lipid bilayer from plastocyanin (Pc) or cytocrome c6 to ferredoxin (Fd) or flavodoxin. PS I is a socalled Type I photosynthetic reaction center, which employs low-potential iron−sulfur clusters (FA/FB) as the terminal electron acceptors. The three-dimensional structure of PS I from the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus has been solved by X-ray diffraction to a resolution of 2.5 Å.20 The monomeric PS I complex contains 12 different protein subunits, nine of which are membrane-bound and three of which are membrane-extrinsic. The membraneembedded core of PS I is a heterodimer of the two largest subunits, PsaA and PsaB, which along with PsaF, PsaI to PsaM, and PsaX binds 96 molecules of Chl a, 22 molecules of βcarotene, two molecules of phylloquinone (PhQ), and an interpolypeptide [4Fe−4S] cluster FX. The terminal [4Fe−4S] clusters, FA/FB, are bound to the peripheral stromal subunit, PsaC. The arrangement of the electron transport cofactors in the PsaA, PsaB, and PsaC subunits is shown in Figure 1. The primary electron donor, P700, is comprised of two Chl a molecules. The spectroscopically detected PS I cofactors include P700 (the special pair of molecules Chl1A and Chl1B), A0 (one or two pairs of Chl molecules denoted as Chl2A/Chl3A and Chl2B/Chl3B), A1 (one of the two molecules of phylloquinone (A1A/A1B)), and FX, FA, and FB (the three [4Fe−4S] clusters) (see Figure 1). According to the currently accepted paradigm, the primary electron donor P700 is promoted to the excited singlet state P*700

Figure 1. Placement of electron-transfer cofactors in PS I from Thermosynechococcus (T.) elongatus20 as derived from the crystallographic coordinates given in the Protein Data Bank (PDB) entry 1JBO. The potential electron-transfer pathways are indicated by arrows. The characteristic times of subsequent electron transfer steps are given for room temperature.

after absorbing a quantum light. This event is followed by the primary act of charge separation between P700 * and the primary •+ •− acceptor A0, forming the radical pair P700 A0 . At room temperature, the electron is then transferred to the adjacent A1 within 50 ps, then to the iron−sulfur cluster FX in ≤200 ns, and further to FA/FB. Alternative models for the first steps of charge separation have been suggested; however, they all •− propose the radical pair P•+ as the first stable radical 700A1 21−23 pair. In the absence of an exogenous electron acceptor, the •− electrons on F•− recombine with P•+ X and [FA/FB] 700 with lifetimes of 0.5−5 ms and 30−100 ms, respectively (for a review, see ref 24). The pseudosymmetric arrangement of the cofactors, as well as the convergence of the A- and B-branches at the FX cluster, suggests that both branches may be active in electron transfer. However, the issue of whether electron transfer in PS I utilizes the A-branch of cofactors, the B-branch of cofactors, or both branches has been a topic of lively debate for the past decade.25 •− At temperatures below 200 K, the lifetime of the P•+ 700A1 26 radical pair increases to more than 20 μs, allowing orientation resolving dipolar EPR experiments to be performed on this radical pair intermediate. The prime objective of the present study was to obtain the structural parameters which would enable one to make a judgment about the participation of Aand B-branches of cofactors in electron transfer. During the work, however, additional unexpected features of the system were found. In the following we report our findings and compare them with the results of previous EPR investigations.

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EXPERIMENTAL SECTION Materials. The samples were prepared from the cells of the wild-type cyanobacterium Synechocystis sp. PCC 6803 adapted to D2O by subsequent increase of the D2O/H2O concentration as described elsewhere.27 The concentration of Chl a in PS I samples was about 5 mM for Q-band EPR and 10 mM for Wband EPR experiments. Prior to the EPR measurements a B



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Figure 2. Left: Microwave (mw) pulse irradiation schemes of various EPR techniques utilized in this work. The upper panel of the figure shows inphase detection methods. The dipolar EPR methods with out-of-phase echo detection are shown in the lower panel. For the meaning of the acronyms, see text. Right: The panels show examples of the time-domain recordings for W-band ESEEM, RIDME, and PELDOR experiments. Blue and red lines show out-of-phase and in-phase echo decay traces, respectively. For details, see Supporting Information.

laser flash to allow for subtraction of stationary background signals. W-Band EPR Experiments. W-band EPR measurements were performed on a home-built 95 GHz/3.4 T multipurpose EPR spectrometer described previously.29,30 The spectrometer is equipped with a TE011 optical transmission cavity. The laser light was guided to the center of the cavity through a quartz fiber of 0.6 mm diameter. The heterodyne mw bridge allows us to perform cw, transient, and multifrequency pulsed EPR experiments. The TREPR spectra were recorded using the direct-detection method without field modulation. For the pulsed EPR measurements a time delay after the laser flash, TDAF, was introduced when applying various mw pulse sequences for echo generation (see Figure 2). The typical settings for pulsed experiments were: ESEEM: tp(π/3) = 20 ns, τ0 = 50 ns, Δτ = 20 ns; RIDME: tp(π/2) = 30 ns, T = 30 μs, τ0 = 50 ns, Δτ = 10 ns; PELDOR: tp(π/2, νa) = 30 ns, T′ = 1.2 μs, T = 3.6 μs, τ0 = 50 ns, Δτ = 10 ns. The length of the pump mw pulses in the PELDOR sequence was determined for each experiment separately taking into account the bandwidth of the overcoupled mw cavity and the frequency difference, νa − νb.18 For νa − νb ≈ 170 MHz this pulse length was tp(π, νb) ≈ 300 ns. In all experiments the phase of the first mw pulse was cycled between +x,−x, which is a general procedure in pulsed magnetic resonance.31 The pulse sequences were repeated with the 3 Hz repetition rate of the Nd:YAG laser (532 nm, 1 mJ on the sample surface) using TDAF = 500 ns. The single echo-response trace was averaged four times (two for each of +x,−x phase settings) in the ESE, ESEEM, and RIDME experiments and eight times (four for each +x,−x phase

solution of sodium ascorbate (final concentration, 5 mM) was added to the sample to reduce potentially oxidized cofactors. The sample solution was transferred to EPR tubes of ID = 1.6 mm for Q-band EPR and 0.6 mm for W-band EPR. After 5 min dark incubation at 4 °C, the sample was frozen in the dark in liquid nitrogen and subsequently transferred into the precooled EPR cavity. Q-Band EPR Experiments. Q-band transient EPR •− measurements of the spin-polarized radical pair P•+ 700A1 were carried out using a Bruker ER200E spectrometer equipped with a Bruker ER051QG microwave (mw) bridge using the directdetection method without field modulation (TREPR). A homebuilt TE011 Q-band cavity was used.28 In the resonator wall 12 horizontal slits of 0.3 mm width were machined to allow for in situ light excitation of the sample (65% light transmission). Pulsed light excitation at 532 nm was achieved with the Vibrant 355 II Laser system from OPOTEK. The light was coupled into the cavity by an optical fiber with an output energy of 7 mJ. Pulsed Q-band EPR experiments were carried out on a Bruker ELEXSYS E580 spectrometer with a Super Q-FT mw bridge equipped with a home-built ENDOR cavity similar to that used for TREPR measurements. Field-swept echo-detected EPR spectra were recorded using a two-pulse spin echo sequence in which the echo intensity was recorded as a function of the external magnetic field. Microwave pulses of 40 ns (π/2) and 80 ns (π) with interpulse spacing τ = 400 ns were used. Pulsed light excitation was achieved using a frequency-doubled GCR-130 Nd:YAG laser system from Spectra Physics (532 nm, 8 ns, up to 10 mJ in front of the EPR cavity). The repetition rate was set to 3 Hz. The pulse sequence was repeated twice with 1 μs before the laser flash and TDAF = 1 μs delay after the C



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settings) in the PELDOR experiment. The PELDOR sequence was repeated twice with the pump mw pulse switched on and off, which allowed for the correction of the background RIDME and ESEEM contributions if necessary. The quadraturedetected echo traces (s−y, in-phase; sx, out-of-phase) were digitized with 500 Megasamples/s (2 ns per point) by a digital oscilloscope and transferred to the computer for further evaluation. The procedure of data analysis was similar to that reported previously18 and illustrated in the Supporting Information. All computation routines were programmed and executed using the EasySpin toolbox for the Matlab program package.32,33 It is noted that the PELDOR pulse sequence for the stimulated spin−echo detection utilized in this work differs from the three-pulse PELDOR version first introduced by Milov et al.14,15 It also differs from the widely used four-pulse constant-time PELDOR sequence at X-band introduced by Jeschke et al.34 There are two reasons for choosing the stimulated spin−echo-based PELDOR pulse sequence. (i) The mechanism of the out-of-phase echo formation in spincorrelated radical pairs (SCRPs) requires that the evolution of the magnetization in τ-space is as short as possible. (ii) The low mw power available at the W-band results in the requirement of long pump mw pulses for large frequency differences in the two-frequency PELDOR experiment. This problem can be solved by using dual-mode mw cavities at Wband, as was demonstrated recently.35 To provide an absolute magnetic field calibration, to ensure the sweep linearity over the magnetic field range in question, and to enable a precise setting of the magnetic field value in the PELDOR experiments, a standard sample (Mn2+ in MgO) was used as a field marker.36

•− Figure 3. (a) Arrangement of the cofactor radicals P•+ 700 and A1 within PS I. (b) Geometrical representation of the respective g-tensor frames of A(xA,yA,zA) and P(xP,yP,zP) in terms of the polar angles ηA, ϕA and ηP, ϕP and the dipolar vector rAP. The angle ζAP defines the relative tilt of the A- and P-frames around the dipolar axis. The inset shows the gvalue selection in the A-frame of reference by the external magnetic field.

νAP =

(2)

where θ is the angle between the Zeeman field B0 and the pair axis director and νdd = μ0·gA·gP·μ2B·(4πh·r3AP)−1 is the principal •− dipolar frequency. For gA ≈ gP ≈ ge (fulfilled for P•+ 700A1 ), it is 3 given by νdd = 52.04 MHz·(r0/rAP) when choosing r0 = 1 nm. Thus, the electron−electron coupling tensor in frequency units in its principal axes frame can be written as

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⎞ ⎛ νdd ⎟ ⎜ νdd D=⎜ ⎟ ⎟ ⎜ −2νdd ⎠ ⎝

THEORY In this section, we briefly summarize the definitions of the radical pair geometry and the basics of dipolar EPR spectroscopy. A more extended description can be found in our previous publication.18 •− The radical pairs P•+ 700A1 are fixed in a solid matrix (protein scaffold) at a certain distance and orientation to each other (see Figure 3a). To define the position of the radicals in the pair, the respective coordinate frames, A- and P-frames, are attached to the radical moieties as follows: (i) The directions of the principal axes of the A- and P-frames of reference coincide with the principal directions of the corresponding g-tensors. (ii) The origins of the A- and P-frames are set to the electron spindensity centers of the respective radicals. Since the distance, rAP, •+ between A•− 1 and P700 well exceeds the extension of their spincarrying orbitals, the point-dipole approximation with the spins fixed at the spin-density centers adequately describes the dipole−dipole interaction between the electron spins. In the point-dipole approximation, the contribution of the electron− electron dipole interaction to the spin Hamiltonian is given by

(3)

where ν(θ = 0) = ν∥ = −2νdd and ν(θ = 90°) = ν⊥ = νdd are called principal parallel and perpendicular dipolar frequencies, respectively. The g-values gA(θA,φA) and gP(θP,φP) in eq 2 refer to the radical pair selected by the magnetic field B0, as is explained for the A-frame in the inset in Figure 3b. The orientation selection by the magnetic field is given by the well-known relation (which acts as an orientation selector) 2 g i (θi , φi)2 = (gxx cos2 φi + g yy2 sin 2 φi) ·sin 2 θi + gzz2 cos2 θi

(4)

where gxx, gyy, and gzz are the principal components of the gtensor of the corresponding radical with index i = A, P. The corresponding dipolar frequency is given by eq 2 with cos θ = sin θi sin ηi(cos φi cos ϕi + sin φi sin ϕi) + cos θi cos ηi

HDD = SA⃗ ̂ ·D·SP⃗ ̂ =−

μ0 gA ·gP ·μB2 · ·(1 − 3 cos2 θ ) = νdd ·(1 − 3 cos2 θ ) 3 4πh rAP

(5)

calculated as the angle between unit-length magnetic field director and director of the pair axis with respect to the corresponding A-, P-frames. The relative placement of the A- and P-frames is defined by the distance between their origins, rAP, and five independent angles. Typically, the two polar angles, ηA and ϕA, are used to define the direction of the pair axis in the A-frame, while the

⎛ 3(S ⃗ · r ⃗) ·( r ⃗·S ⃗ ) S ⃗ ·S ⃗ ⎞ P ·gA ·μB ·gP ·μB ·⎜ A 5 − A3 P ⎟ 4π ℏ rAP rAP ⎠ ⎝ μ0

(1)

The dipole coupling frequency is defined by D



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•− Figure 4. (a) W-band and (b) Q-band transient EPR spectra of P•+ 700A1 in the deuterated PS I from the cyanobacterium Synechocystis sp. PCC 6803 recorded at 120 K. TREPR (upper trace) and field-swept ESE-detected EPR (lower trace) spectra were recorded 1 μs after repetitive laser flashes (3 Hz). Field-swept ESE-detected EPR spectra are corrected for background signals by using the spectrum obtained 1 μs prior to the laser flash. The dashed lines give the respective calculated SCRP EPR spectra considering only A•− 1 contributions. A and E stand for absorption and emission. On the top, the corresponding calculated EPR spectra of thermalizated A•− 1 are shown in first-derivative representation.

three Euler angles, α, β, and γ, define the orientation of the Pframe with respect to the main (observer) A-frame, or vice versa when choosing as the mainframe of reference the P-frame. In dipolar EPR spectroscopy, where the frames of both radicals are used as observer frames, it is more convenient to use the “symmetrical angle system”,18 i.e., two polar angle sets, ηP,ϕP and ηA,ϕA, defining the direction of the dipolar (pair) axis in both frames, and the angle ζAP which gives the relative rotation of both frames around the dipolar axis (see Figure 3b). The high-field dipolar EPR methods are divided into different types according to the degree of magnetoselection: Single magnetoselection methods, for instance RIDME, operate at one fixed microwave frequency/magnetic field value; i.e., they select gA(θA,φA) = gP(θP,φP) and yield dipolar coupling frequencies of each radical in the pair separately. Thus, the cos θ value in eq 5 can be obtained from the measured dipolar frequency at each magnetic field position (see eq 2) within the radical’s EPR spectrum, assuming that the longitudinal relaxation times are not orientation dependent. As result, the polar angles η and ϕ are only obtained from the g-values at selected field position and cos θ (see eqs 4 and 5). PELDOR, as a double magnetoselection method, probes different resonant positions within the EPR spectrum of each radical in the pair, i.e., gA(θA,φA) ≠ gP(θP,φP), and dipolar responses are only detected if gA(θA,φA) and gP(θP,φP) belong to the same radical pair. Only this method is capable of delivering all the six parameters that define the full radical pair geometry.

reported data. The details are given in the Supporting Information. In the following sections, we describe orientation-resolving dipolar high-field EPR experiments and data analysis which are based on the protocol we had previously developed for dipolar •− high-field EPR experiments on the transient P•+ 865QA radical 18 pair in bacterial RCs. It includes a step-by-step analysis of TREPR spectra of SCRP-polarized radical pairs as well as the analysis of RIDME and PELDOR experimental results. For PS I, the protocol was modified owing to the considerable differences between the two radical-pair systems. The magnetoselection in the EPR spectrum of P•+ 700 is substantially reduced in comparison to the primary donor P•+ 865 in the bacterial RC due to its smaller g-tensor anisotropy: g(P•+ 700) = [2.00309 2.0255 37,38 Hence, we 2.00227]; g(P•+ 865) = [2.00323 2.00235 2.00196]. strove for precise information about the polar angles (ηA, ϕA) for the quinone cofactor A•− 1 determining the direction of the dipolar vector in the g-frame of A•− 1 . Its larger g-tensor anisotropy provides the desired Zeeman magnetoselection at W-band mw frequencies. Next, we further analyzed the experimental findings to obtain the polar angles (ηP, ϕP) for the primary donor P•+ 700. Orientation of the Dipolar-Axis Vector rAP in the gTensor Frame of A•− 1 . Information about the orientation of the dipolar coupling axis with respect to the g-tensor axes of the A•− 1 cofactor can be obtained from the EPR spectra of the •− 18,39 The transient W-band TREPR spectrum of SCRP P•+ 700A1 . fully deuterated PS I is shown in Figure 4a (upper trace). It agrees well with the TREPR spectrum of deuterated PS I observed previously.40 The integral intensity of the spectrum is •− zero, indicating that the P•+ 700A1 radical pair is generated in a 41 pure singlet state. The low-field spectral region is dominated by the gxx and gyy components of the A•− 1 g-tensor which are completely resolved by W-band EPR. According to the SCRP mechanism, the sign and size of the electron spin polarization is determined by the value of the dipolar coupling when a given principal g-tensor axis is parallel to the external magnetic field (canonical orientation). The dipolar fields projected onto the principal axes of the A•− 1 g-tensor are given by

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RESULTS AND DISCUSSION Prior to performing dipolar EPR experiments, the PS I sample was characterized with regard to its photophysical and photochemical properties using a variety of W-band EPR methods. The essential characteristics are the reversibility of the •− electron transfer, the lifetimes of the P•+ 700 and A1 transient charge-separated states, the lifetime of SCRP spin polarization, and the relaxation dynamics of the cofactor radical ions. These measurements were performed at 120 K. The main characteristics were found to be in good agreement with previously E



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•− Figure 5. (a) Graphical solution for the angles ηA, ϕA in the A-frame as derived for the spin-correlated radical pair P•+ 700A1 . The selection probability •− traces TrA∥ and TrAy/x are plotted on the A1 frame sphere in red and blue, respectively. The part of TrAy/x which has to be excluded from •− consideration due to dxA/d0 < 0 in eq 8 is shown in magenta. For details, see the text. (b) Top: The W-band TREPR spectrum of the SCRP P•+ 700A1 •− is shown for referring to the spectral positions. Bottom: W-band RIDME spectrum of the SCRP P•+ A in frozen solution at 120 K. Shown is the 700 1 contour plot of the positive Fourier amplitudes of out-of-phase detected RIDME traces taken over the whole EPR spectrum of the radical pair. The long mixing period of the pulse sequence is T = 30 μs, and the delay after flash is TDAF = 500 ns.

probability of the orientation (ηA, ϕA) by a Gaussian is given by exp{−[cA(ηA,ϕA) − cA̅ ]2/δcA2},18 where cA̅ and δcA represent the mean values and estimated errors in eq 7. This orientation probability is shown in Figure 5a by the trace marked TrAy/x. Since the sign of the dipolar field dxA is negative, only those orientations in the selection trace TrAy/x have to be considered which are satisfying the condition

dx A =d0·(1 − 3 sin 2 ηA cos2 ϕA ) dy A =d0·(1 − 3 sin 2 ηA sin 2 ϕA ) dz A =−dx A − dy A = d0·(1 − 3 cos2 ηA )

(6)

where d0 = (h/μB·ge)νdd is the principal dipolar field when approximating gA ≈ gP ≈ ge = 2.0023; see eqs 4 and 5. The gxx spectral line of A•− 1 appears in emission. This yields a negative sign of the dipolar field, dxA < 0. The gyy line, on the other hand, appears in the absorption/emission derivative form, i.e., in-phase with the first derivative of the A•− 1 EPR absorption (see top spectrum in Figure 4a). This yields a positive sign of dyA. The intensity ratio of the gxx and gyy lines, as compared to the derivative of the EPR absorption, is determined by the ratio of the dipolar fields at these positions. Thus, from the analysis of the SCRP EPR spectrum the desired information about the (ηA, ϕA) angles can be obtained. To simulate the spectral contributions of A•− 1 according to the SCRP mechanism,9 the exact g-tensor values and the corresponding EPR linewidths are required. These parameters cannot be determined with sufficient accuracy from independent cw EPR experiments on samples with cyclic electron transfer due to the short (95% under our experimental conditions. This is in agreement with the results of our previous investigations on PS I mutants.68,69

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COMPARISON WITH THE X-RAY CRYSTAL STRUCTURE OF PS I In this section we compare the radical-pair geometry parameters obtained in this work by high-field dipolar EPR spectroscopy with the structure of a model reference pair for •‑ which the radical ions P•+ 700 and A1 are assumed to stay at the same positions as their precursor cofactors, P700 and A1A or A1B, in the dark state of PS I. These positions are known from earlier high-resolution X-ray crystallography.20 To describe the positions of the radical ions within the reference-pair coordinate system in terms of length and polar angles of the dipolar axis, the respective reference frames were attached to the radicals to be coaxial with their g-tensors. For both quinones, A1A or A1B, the principal x-axis of the g-tensor was aligned to be collinear with the averaged direction of the CO bonds of the phylloquinones, and the y-axis was chosen to be perpendicular to the x-axis in the plane of the quinone rings. The z-axis was taken to be perpendicular to both x- and y-axes. The origin of the A1 reference frame was fixed at the center of the quinone ring that approximates the electron spin-density center of the radical. This procedure is conventional for quinone molecules and follows from their symmetry. However, any reconstructed geometry of the quinone g-frames suffers from symmetry ambiguity: The π rotations around the principal g-axes generate four possibilities to place the right-handed gtensor coordinate system within each of the quinone molecules. For positioning the g-tensor, a configuration was chosen which allows for the smallest polar angles determining the dipolar director, similar to the selection made previously.40 In such a frame the dipolar director points to the second octant of the reference frame (see Figure 10).

Figure 10. Arrangement of the primary electron donor P700 (the Chl1A and Chl1B special pair) and the phylloquinones A1A and A1B, as based on the X-ray crystal-structure data20 (PDB entry 1JBO). The distances between P700 and A1A in the A-branch, and between P700 and A1B in the B-branch, correspond to distances between the spin-density center in P•+ 700 (for references, see refs 66 and 67) and the geometrical centers of the quinone rings of A1A and A1B.

In contrast to the quinone reference frame, which is connected to the molecular axes, the definition of the P700 reference frame requires exact knowledge of the g-tensor orientation in the molecular axes system of the primary donor. Unfortunately, this information cannot be extracted from the results of the previous studies. All our attempts to obtain a unique (and reliable) g-tensor orientation failed due to L



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CONCLUSIONS In this work we have shown that by combining cw high-field transient EPR at W-band (95 GHz, 3.4 T) with the orientationresolving high-field pulsed dipolar EPR methods RIDME and PELDOR in conjunction with employing an elaborate data analysis, distance and orientation information on light•− generated transient radical pairs P•+ 700A1 in frozen solution of deuterated PS I reaction centers from the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 can be obtained with high accuracy. These dipolar-EPR methods are distinguished by their applicability also to randomly distributed radical pairs with no long-range order; i.e., they do not require single-crystal samples. Details of the three-dimensional molecular structure can be elucidated to a precision comparable to or better than those of high-resolution protein X-ray crystallography. We have applied the RIDME and PELDOR measurements exclusively to weakly coupled spin-polarized •− radical pairs P•+ 700A1 in their spin-correlated state since the radical pairs in their thermally equilibrated Boltzmann state could not be observed owing to the short lifetime of A•− 1 in the cyclic electron transfer. The measured positions and relative orientations of the ion •− pair are compared with previously radicals in the P•+ 700A1 reported EPR geometries and with the 3D-structure of the precursor cofactors P700 and A1 as known from X-ray crystallography. At cryogenic temperature the light-induced cyclic electron transfer was found to proceed exclusively along the protein A-branch of the PS I cofactor chain. The position and orientation of the reduced phylloquinone coincide with those of the precursor phylloquinone molecule revealing that no orientational changes of A1 occur upon charge separation. The structure of P•+ 700 was found to exhibit several conformational substates with slightly different electron-spin density distributions. This causes several distinct orientations of the P•+ 700 g-tensor axes with respect to the molecular frame of the primary donor P700. All quantitative statements given in terms of structure and dynamics are underpinned by discussing the error margins. It is hard to imagine any other spectroscopic or crystallographic method than orientation-resolving high-field PELDOR with similar capabilities of revealing such subtle structural differences of conformational substates. Concerning the data analysis described in this work we want to make the following point: In most laboratories the typical data analysis procedure of pulse dipolar EPR spectroscopy involves spectral-fitting computer routines that simulate the experimental electron-dipole-modulated spin−echo decays on the basis of an approximate spin Hamiltonian. This fitting procedure requires an initial structural model as input data for the relative orientation of the paired radicals. Such input data have to be provided by other independent methods, at best by high-resolution X-ray crystallography. Hence, the data-fitting procedure can only render improvements of the initial input structure and, thus, cannot be used to solve the geometry of a radical pair of unknown structure. The reason behind this is the multitude of degenerate symmetry-related solutions of the structure problem, which principally cannot be distinguished by any magnetic-resonance method. In contrast, in the present work several high-field EPR, RIDME, and PELDOR techniques in combination with analytical and graphical data analysis procedures have been used by which all symmetry-allowed radical-pair geometries were foundand this without fitting

echo-modulated decay traces, i.e., without the need of any a priori information about the radical-pair structure. To exclude solutions which are not compatible with other independent observations, we resort to available X-ray structure information. Our approach does not require any fine details of the X-ray structure but only information about the approximate location of the molecules forming the radical pairs. Other key advantages of high-field PELDOR and related dipolar EPR methods for structure determination are: (i) they can be selectively adapted to specific questions concerning the radical molecules embedded in a protein like their static orientation relative to each other; (ii) they are able to reveal local displacements of protein subunits or cofactors in their binding sites that may accompany functional processes of proteins in action; i.e., they are able to reveal dynamic changes of molecular conformations; (iii) they are particularly suited for characterizing potential structural transformations of protein sites as a result of site-directed mutagenesis. Such information is extremely important for studies of biomolecules with sitespecific nitroxide spin labels and forms the basis of a meaningful application of site-directed spin labeling in structural biology.

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ASSOCIATED CONTENT

S Supporting Information *

Details of the PS I characterization by high-field EPR methods; examples of out-of-phase ESEEM, RIDME, and PELDOR experimental recordings; time-to-frequency domain transformation of experimental out-of-phase recordings; calculation of RIDME spectra; evaluation of P•+ 700 parameters; comparison of calculated TREPR spectra using a multitude of reported structures; results of spin-density molecular-orbital calculations. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0049-208-3063555. Fax: 0049-208-3063951. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Y. Grishin (Institute of Chemical Kinetics and Combustion, Novosibirsk, Russia) for his technical support, M. Plato (Free University Berlin) for clarifying discussions concerning the geometical data analysis and the calculations of primary donor electronic structure parameters, A.A. Dubinskii (Semenov Institute of Chemical Physics, Moscow, Russia) for helpful discussions concerning the dipolar EPR methods and data analysis, A.Yu. Semenov (A.N. Belozersky Institute of Physical-Chemical Biology, Moscow, Russia) for illuminating discussions concerning Photosystem I electron transfer, and M. Antonkine for help with sample preparations. We gratefully acknowledge financial support by the Max Planck Society to A.S., J.N., K.M., and W.L. This work was supported by a grant from the National Sciences Foundation (MCB1021725) to J.H.G.

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REFERENCES

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