Light-Induced Electron Spin-Polarized (ESP) EPR Signal of the P800+

Subscriber access provided by UNIV OF DURHAM

Article

Light-Induced Electron Spin-Polarized (ESP) EPR Signal of the P800 Menaquinone Radical Pair State in Oriented Membranes of Heliobacterium modesticaldum: Role/Location of Menaquinone in the Homodimeric Type I Reaction Center +

–

Toru Kondo, Masahiro Matsuoka, Chihiro Azai, Masami Kobayashi, Shigeru Itoh, and Hirozo Oh-Oka J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b12171 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Light-Induced Electron Spin-Polarized (ESP) EPR Signal of the P800+ Menaquinone– Radical Pair State in Oriented Membranes of Heliobacterium modesticaldum: Role/Location of Menaquinone in the Homodimeric Type I Reaction Center Toru Kondoa†*, Masahiro Matsuokab, Chihiro Azaib‡, Masami Kobayashic, Shigeru Itoha, and Hirozo Oh-okab*

a

Division of Material Science (Physics), Graduate School of Science, Nagoya University,

Furocho, Chikusa, Nagoya 464-8602, Japan. bDepartment of Biological Sciences, Graduate School of Science, Osaka University, Osaka 560-0043, Japan. cDivision of Materials Science, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan.

†

Present address: Department of Chemistry, Massachusetts Institute of Technology,

Cambridge, MA 02139, USA ‡

Present address: Department of Bioinformatics, College of Life Sciences, Ritsumeikan

University, Shiga 525-8577, Japan

*

Corresponding authors:

(T. K.) Phone: +1-617-452-2628; E-mail: [email protected] (H. O.) Phone: +81-6-6850-5424; E-mail: [email protected]

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Function/location of menaquinone was studied in the photosynthetic reaction center of Heliobacterium (Hbt.) modesticaldum (hRC), which is one of the most primitive homodimeric type I RC. The spin-polarized electron paramagnetic resonance (ESP-EPR) signals of light-induced radical pair species, which are made of oxidized electron donor bacteriochlorophyll g (P800+) and reduced menaquinone (MQ–) or iron sulfur cluster (FX–), were measured in the oriented membranes of Hbt. modesticaldum at cryogenic temperature. The spectral shape of transient ESP signal of P800+FX– radical pair state varied little with respect to the direction of the external magnetic field. It suggested a dominant contribution of the spin evolution on the precursor primary radical pair P800+A0– state with the larger isotropic magnetic exchange interaction J than the anisotropic dipole interaction D. The pure P800+MQ– signal was simulated by subtracting the effects of spin evolution during the electron transfer process. It was concluded that the J value of the P800+MQ– radical pair is negative with an amplitude almost comparable to |D|. It is in contrast to a positive and small J value of the P700+PhyQ– state in PS I. The results indicate similar but somewhat different locations/binding sites of quinones between hRC and PS I.


Page 2 of 24

Page 3 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction The primary photoreactions in photosynthetic organisms occur in the reaction center pigment-protein complexes (RCs). Although RCs vary somehow among photosynthetic organisms, all the known RCs are comprised of two identical or homologous polypeptides that bind multiple electron transfer cofactors and light harvesting pigments. They can be classified into four major types. The one is the homodimeric type I RCs of anaerobic bacteria, and the others are the heterodimeric RCs: photosystem I and photosystem II (PS I and PS II) RCs of plants and cyanobacteria and the type II RCs of anaerobic/aerobic purple photosynthetic bacteria. The structures of the heterodimeric RCs have been determined by X-ray crystallography.1-5 The structure of homodimeric type I RC of green sulfur bacteria (gRC) is still unclear. However, very recently, Gisriel C. et al. reported the structure of homodimeric type I RC of heliobacteria (hRC) that showed high similarity to the PS I RC.6 The hRC has been assumed to be one of the oldest type I RC that remains the feature of the ancestral RC.7-9 Therefore, the study of the function/structure of hRC will give us a clue to understand the evolution of the present RCs. Especially, quinone functions, which differ significantly between the type I and type II RCs,10-14 would be the important key factors for the differentiation of RCs. Quinones are known to serve as the secondary electron acceptors in all the heterodimeric RCs including PS I. In hRC and gRC, however, the quinone functions have not been identified.15-16 The hRC has been assumed to contain five (or six) electron transfer cofactors, that is, P800, A0, (A1 if we include menaquinone, MQ), FX, and FA/FB similar to those in PS I.15-16 The primary electron donor P800 is a special pair of BChl g’ as a counterpart of P700, which is a pair of Chl a and Chl a’, in PS I.11,17-18 The primary electron acceptor A0 is a derivative of chlorophyll a, 81-hydroxy-Chl a (81-OH-Chl a), similar to A0 in PS I that is a monomeric Chl a.11,19-20 The terminal electron acceptors FX, FA, and FB are [4Fe-4S] type iron-sulfur clusters as in PS I.21-24 FX– represents the S = 3/2 spin state22,25 (our previous assignment of the FX– signal with a spin state of S = 1/226 seems to come from an artifact due to the modification of spin saturation).24 The locations of P800, A0 and FX were identified in the recently-revealed hRC structure.6 However, no MQ was identified in the structure. It



is, therefore, suggested whether MQ was dissociated during the hRC preparation procedure or does not function as the electron carrier in hRC.6 PS I contains the tightly-bound two phylloquinone (PhyQ) molecules that function as A1 to accept electrons from A0.10,12,27 Isolated hRC also contains menaquinone-9, while the MQ content fluctuates between zero6 and 1.4-1.628-29 among the hRC preparations, suggesting the loose binding or its diffusive migration between hRC and the Q-pool in membranes.30 The evidence for the function of MQ as A1 in hRC has long been scarce31-32 or even negative.6,33-37 However, we recently detected a new spin-polarized electron paramagnetic resonance (ESP-EPR) signal of the P800+MQ– radical pair state in a hRC core complex (hRCc) isolated from Heliobacterium (Hbt.) modesticaldum, which contains FX and MQ but not FA/FB.25 The hRCc preparation was estimated to contain ~3.9 MQ molecules per RC (see Supporting Material). Almost the same P800+MQ– signal was detected in the membrane preparations.25 It was observed only after the pre-reduction of FX, and eliminated after the diethyl ether treatment of lyophilized hRC that selectively extracted MQ.25 The ESP signal exhibited a wide spectral shape extending to the high g-value range, suggesting the magnetic interaction between P800+ and MQ–. These results can be interpreted if MQ in hRC accepts an electron from P800 fast as does PhyQ in PS I. On the other hand, this P800+MQ– signal exhibited an A/E-type (A, absorption; E, emission) ESP pattern in contrast to the E/A/E pattern known for the P700+PhyQ– ESP signal in PS I.38-40 In PS I, the primary photoreaction produces the radical pair state P700+A0– first, and then P700+PhyQ– and P700+FX–. The latter two retain the ESP features because of the faster electron transfer processes than the spin-depolarization occurring on a time scale of a few tens of microseconds. The ESP patterns are mainly contributed by three factors (see Supporting Material for details); (1) the g-values and g-tensor orientations of the component radicals, (2) relative arrangement of molecules forming the radical pair, i.e. the magnetic dipole (D) and spin exchange (J) interactions, (3) the spin evolution during the stay on precursor radicals.36 The A/E pattern of P800+MQ– ESP signal was not interpreted based on the assumption that the factors (2) and (3) in hRC are identical to


Page 4 of 24

Page 5 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


those in PS I.25 It suggested the arrangement, magnetic properties, and spin dynamics during the electron transfer to MQ molecule in hRC to be different from those of PhyQ in PS I. The electron transfer time from A0 to MQ and FX in hRC has been assumed to be significantly different from the corresponding ones in the A0→PhyQ→FX in PS I. The two steps in PS I occur with time constant (tc) of 30 ps and 15-200 ns, respectively, at room temperature.10 However, A0– was re-oxidized with a tc of 600 ps in hRC, i.e. 20 times slower than the corresponding step in PS I.35,41 Moreover, the ESP signal of P800+MQ– was detectable only after FX– was pre-accumulated.25 This implies that MQ– should reduce FX with a tc around 600 ps if MQ works as A1.25,35 Then, the evolution of spin state during the stay on the long-lived P800+A0– radical pair would strongly affect the ESP patterns of both P800+MQ– and P800+FX–36,42 although the magnetic property of P800+A0– has been ambiguous so far. The ESP signal in hRC, thus, suggests the unique electron transfer process in hRC, and will help us to understand the function/location of MQ in hRC. In this study, we prepared the oriented sheets of membranes isolated from Hbt. modesticaldum, and carefully measured the angular dependencies of the flash-induced ESP signals on the external magnetic field. The analysis of the results suggested that the P800+MQ– and P800+FX– states were produced in the sequential electron transfer from the P800+A0– state. Another possibility that MQ does not function in hRC core as suggested from the struture6 was also tested.

2. Materials and Methods 2.1. Sample preparations A strain of Hbt. modesticaldum was generously provided by M. T. Madigan (Southern Illinois University, Carbondale, IL). Cells were grown anaerobically in a PYE medium in a 1 L bottle under continuous illumination with tungsten lamps.43 To avoid the accumulation of lysed cells in the late-logarithmic growth phase, cultivation was performed at 47 °C only for 18-20 h using 1% inocula.26



All the procedures for the preparations of membranes as well as the EPR measurements were carried out under anaerobic conditions, as previously described.44 All the media were fully degassed and flushed with N2 gas before being used. The cells were harvested by centrifugation at 12,000 g for 10 min, suspended in 7-8 mL of buffer A [50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 2 mM DTT], and disrupted by being passed through a French pressure cell three times at 20,000 psi. After removal of the cell debris by centrifugation at 12,000 g for 10 min, the membranes were collected by ultracentrifugation at 180,000 g for 1 h, and suspended in buffer B that contained 200 mM glycine-KOH (pH 10.0), 1 mM EDTA, and 2 mM DTT. After ultracentrifugations, the membranes were directly pasted on thin polyester sheets to prepare oriented membranes (see below). The number of quinone molecules included in cells, RC core, and highly-purified RC core preparations was estimated to be about 20-30, 3.9 and 0.8 per RC, respectively, based on the HPLC (see Supporting Material and Figure S1). Thus, the membrane preparation should contain ~3.9 MQ/RC at least. Membrane fragments of spinach PS I were prepared as described in ref.45

2.2. Oriented membranes Photosynthetic membranes have been oriented either by stacking as multi-layers46-47 or by aligning under the external magnetic field.48-49 In the present study, we prepared multi-layers of oriented membranes as described by Mino et al.46 The membranes isolated from Hbt. modesticaldum were spread on thin polyester sheets. The sheets were then dried in the dark for 5-7 hours at 4 oC by flushing N2 gas that was supplied through the buffer containing dithionite. Several strips of the sheets were layered in parallel in an EPR tube and frozen in the dark. In the measurements, the layers of sheets in the tube were set to be perpendicular to the external magnetic field first. The angular dependent EPR signal of the well-oriented Rieske iron-sulfur cluster was utilized to estimate a correct angle between the membrane normal and the external magnetic field.24 The orientation of an iron-sulfur cluster was also used to standardize the angle in the oriented membranes of PS I.


Page 6 of 24

Page 7 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.3. EPR measurements Transient EPR measurements were carried out using a Bruker ESP-300E X-band spectrometer with 100 kHz field modulation (Bruker Biospin, Germany) equipped with a liquid-helium flow cryostat and a temperature control system (CF935, Oxford Instruments, Oxford, UK). Continuous white light illumination was given from a 500 W tungsten lamp through heat-cut glass filters. A white xenon flash light with the duration time of 10 µs at nearly saturating intensity (X-50, Sugawara Laboratories Inc., Kawasaki, Japan) was given at 1 Hz through a 1 m glass fiber light guide for the measurements of the transient EPR signal. The decay kinetics of signal was measured at varied magnetic fields, and the intensity (at around 10 µs) just after the flash excitation was plotted to obtain the orientation of ESP signal as described previously.25 The observation of the P800+MQ– ESP-EPR signal requires the pre-reduction of FX–.25 In order to accumulate FX–, the hRCc, which was non-oriented, was illuminated at 210 K for 1 h and then cooled to 5 K under illumination in the presence of several milligrams of dithionite as described in the previous report.25 The oriented heliobacterial membranes were also incubated in the dark at room temperature for 5 min with the same amount of dithionite in a buffer containing 100 mM glycine-KOH (pH 10.0), 1 mM EDTA, and 2 mM DTT, and followed by the illumination at 210 K for 1 h and the cooling to 5 K under illumination. Hereafter the pre-illuminated samples were designated as “light-cooled hRC”, and the samples cooled down to 5 K in the dark where the FX remains oxidized were designated as “dark-cooled hRC”.

3. Results and discussion 3.1. Detection of the ESP-EPR spectra of P+Q– and P+FX– radical pairs in PS I and hRC Figure 1 shows the first-derivative spectra of the X-band ESP-EPR measured in PS I (A and B) and hRC (C and D). In PS I, the P700+PhyQ– ESP state can be induced by the flash excitation at



temperatures lower than 200 K due to the suppression of the electron transfer from PhyQ to FX (Figure 1A), and the P700+FX– ESP could be detected at the higher temperatures (Figure 1B).40 The condition for the observation of the P+Q– ESP signal in PS I was significantly different in hRC. In hRC the flash excitation of the dark-cooled hRC induces the P800+FX– ESP signal even at 10 K (Figure 1D) showing the light-induced electron transfer from P800 to FX even at cryogenic temperatures.25,32-33 Only in the light-cooled hRC, where reduced FX was pre-accumulated, the P800+MQ– ESP state was detected as shown in Figure 1C.25

3.2. Angular dependencies of ESP-EPR spectra of P700+PhyQ– and P700+FX– in oriented PS I Both the ESP patterns of P700+PhyQ– and P700+FX– radical pairs in PS I varied depending on the angle between the membrane normal and the external magnetic field. The P700+PhyQ– signal showed a typical E/A/E pattern in the non-oriented membranes (Figure 1A, upper, note that signals were measured and expressed as the derivative types).38-40 When the oriented membranes were set at 0o, an E/A type ESP was observed (Figure 1A, middle). On the other hand, at 90o the spectrum showed the opposite A/E pattern (Figure 1A, bottom). The P700+FX– signal, which exhibited an A/E/A pattern in the non-oriented membranes (Figure 1B, upper), showed A/E and E/A pattern ESPs when measured at 0o and 90o, respectively, in the oriented membranes (Figure 1B, middle and bottom, respectively). Features of the ESP patterns were summarized in Table 1. The ESP pattern of radical pair is known to be described by a linear combination of three terms; singlet, net polarization, and multiplet polarization terms (see Supporting Material).36,42 The first singlet term can be calculated from the magnetic property and component arrangement of the radical pair. The latter two terms, on the other hand, are derived from the spin dynamics on precursor radical pairs that change the population of spin states on the terminal radical pair. The contributions of these terms to the ESP signals were summarized in Table 1. It has been known in PS I that P700+PhyQ– is derived from the primary photoproduct P700+A0– with a very short time constant tc of 30 ps, much faster than the singlet-triplet mixing.36,42 Therefore,


Page 8 of 24

Page 9 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the ESP pattern of P700+PhyQ– is expected to be slightly affected by the spin evolution process due to the fast electron transfer rate. The pattern, thus, has been successfully reconstituted by considering only the singlet term according to the correlated coupled radical pair (CCRP) model (Figure 1A, black solid lines).50 From the spectral simulation, the exchange interaction J and dipole interaction D were estimated to be 0.0006 ~ 0.0046 and –0.20 ~ –0.17 mT, respectively,51-55 indicating the predominance of contribution of anisotropic D compared to that of the isotropic J (|D| > |J| situation). This interprets the variation of ESP pattern depending on the angle of external magnetic field. For the ESP spectrum of P700+FX–, contribution of singlet term should be negligible due to the fast relaxation of spins on the FeS cluster.38,42 Then, the ESP pattern is mainly contributed by net and multiplet polarization terms of the precursor, especially the P700+PhyQ– state that has the dwell time longer than that of the preceding precursor P700+A0– state.42 The dominant contribution of P700+PhyQ– state with |D| > |J|, thus, interprets the angular dependence of ESP pattern of P700+FX– state as shown in Figure 1B.

3.3. Angular dependence of P800+MQ– and P800+FX– ESP-EPR spectra in hRC In contrast to the prominent angular dependency of the ESP signal of P700+PhyQ–, the ESP signal of P800+MQ– in hRC showed only weak angular dependency (Figure 1C). It might come from the long lifetime (600 ps) of the precursor P800+A0– state, which is 20 times longer than that of P700+A0–.35,41 In this situation, the spin polarization state is significantly time-developed in the precursor P800+A0– state, and hence the contribution of the net and multiplet polarization terms to the ESP pattern of P800+MQ– can be no longer ignored. Actually, the spectral simulation based only on the singlet term never reproduced the ESP pattern.25 Therefore, we need information about spin dynamics in the precursor P800+A0– state too in order to analyze the P800+MQ– ESP pattern. The spin polarization developed on P800+A0– should also affect the P800+FX– ESP pattern predominantly because of the fast electron transfer from A0– to FX, either via MQ25,35 or directly.6 It was supported by the result of spectral simulation, where the experimentally obtained P800+FX– ESP



spectrum was almost reproduced with the D and J parameters of P800+A0– at –0.34 and 0.5–1.1 mT, respectively, even with no contribution of the precursor P800+MQ– state.36,42 The estimation of |D| < |J| condition interprets the negligible angular dependence of the P800+FX– signal in Figure 1D. Therefore, the P800+FX– ESP signal in hRC is concluded to be mainly contributed by the precursor P800+A0– state, and indicates the contribution of the net and multiplet polarization terms to the spectrum (Table 1). It is, thus, assumed that the subtraction of an appropriate extent of P800+FX– ESP signal from the P800+MQ– ESP signal will practically cancel the contribution of the P800+A0– precursor state, i.e. the net and multiplet polarization terms (see Supporting Material for the detail of the procedure), and will help the better simulation of the P800+MQ– ESP signal with the singlet term even if the properties of P800+A0– are not given in advance. In addition, it will remove the residual P800+FX– ESP signal arising from the hRCs with the oxidized FX that remained due to the insufficient pre-reduction treatment. This procedure, as well as the angular-resolved measurement, progressed the simulation of ESP spectra as below.

3.4. Simulation of P800+MQ– ESP signal The spectra after the subtraction were shown in upper panels in Figure 2. The calculated ESP signal exhibited the E/A pattern in the non-oriented membranes (Figure 2A, solid line in upper panel). Similar E/A patterns are seen in the oriented membranes too both at 0o and 90o with different amplitudes (Figure 2B and C, solid lines in upper panels). The lower panels in Figure 2 represent the simulated ESP spectra by assuming the same orientation of MQ as that of PhyQ in PS I, where the gx axis of PhyQ is parallel to the dipolar axis Zd connecting P700+ and PhyQ− (gx // Zd). In the simulation, the g-tensors, mutual arrangement, and magnetic dipole interaction D (= –0.17 mT) between P800+ and MQ– were set the same as those assumed in the previous study.25 It is seen that the variation of magnetic exchange interaction J, i.e., various ratios of D and J, modifies the ESP pattern and its angular dependence significantly. The ESP pattern shows strong angular dependence at J = 0 mT as


Page 10 of 24

Page 11 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shown in P700+PhyQ– in PS I (Figure 1A). The simulated signal, however, does not agree with the experimental one. On the other hand, the simulation at J = –0.2 mT and D = –0.17 mT (|D| ≈ |J|) gave the best agreement. In this case, the balance between anisotropic D and isotropic J interprets the weak angular dependence. Instead of the high |J| value as estimated above, low |D| values may also realize the |D| ≈ |J| situation. If we assume the small |D| value, which is comparable to the |J| value of 0.0006 ~ 0.0046 mT as estimated for the P700+PhyQ– state in PS I,54-55 the distance between P800 and MQ in hRC should be 3.3 times longer than 25 Å between P700 and PhyQ. Such a long distance is rather unrealistic and will give the extremely low electron transfer rate between P800 and MQ. It is thus inconsistent with the observation of the rapid decay of the P800+MQ– ESP signal.25 Effect of orientation of MQ was also tested in Figure 3, in which spectra of the P800+MQ– ESP signal were simulated at J = –0.2 mT at varied orientations of MQ molecule. The simulation at gx // Zd, the same orientation as that of PhyQ in PS I, better reproduced the experimental ESP pattern and its angular dependence (Figure 3B). Meanwhile, the simulations at gz // Zd also showed similar spectra (Figure 3D). Therefore, we can expect the quinone-orientation somewhere between gx // Zd and gz // Zd. Further study by the high-frequency EPR and the structural determination of MQ-preserving hRC will give more precise location and orientation of functional MQ.

3.5. Different environments of quinones that may give different J values in PS I and hRC The results in this study estimated the P800+MQ– state to have a negative large J value comparable to D value based on the analysis of ESP signal. The estimated J value is different from the positive small J values of +0.0006 and +0.0046 mT of the P700+PhyQ– state on A- and B-branches, respectively, in PS I.54-55 The J property is known to reflect the overlap between the electronic orbitals of components of the radical pair, including the through-space and through-bond type overlaps that affect the energy gap between singlet and triplet spin state.56 If the contributions of these two overlaps are balanced and cancelled out each other, i.e. in-phase and anti-phase contributions are averaged to



be zero, then the triplet state can be slightly energetically lower than the singlet state, yielding a positive J, because only the exchange repulsion for the singlet spin pair remains.56 This seems to explain the small positive J value seen in P700+PhyQ– in PS I. The distance between P700+ and PhyQ– in B-branch (24.3 Å), which is a little shorter than that in A-branch (25.4 Å)55 may give the larger orbital overlap and the larger exchange repulsion to make J a little more positive. Thus, the J value will be sensitive to the molecular position. Additionally, the through-space and through-bond overlaps can be significantly perturbed by the molecular orientation and binding motif in the binding site. Therefore, the cancellation between the in-phase and anti-phase overlaps as in PS I would be rather fragile and may not be realized in hRC. This situation would lead to the negative J value. The arrangement of MQ as well as the property of the binding site in hRC seem to be different from those of PhyQ in PS I.25-26 Such deviations would disrupt the balancing condition of orbital overlaps as in PS I, and accordingly would give the large negative J value in P800+MQ– in contrast to the situation of P700+PhyQ– in PS I. The electron transfer system in hRC, thus, can be assumed to have the larger |J|, i.e. stronger electronic interaction between P800 and MQ. The high charge recombination rate between P800+ and MQ– observed even at cryogenic temperature in hRC25 might also be interpreted with the large |J| value, which reflects the tunneling matrix element between the electron donor and accepter and determines the electron transfer rate.57-59 The value of exchange interaction J of radical pair can be expressed by the empirical function of J = J0 e–β r, where J0 is an appropriate constant, r is a distance between the radical molecules, and β is a decay constant of the electronic coupling.59-62 β can be related to the overlap of electronic orbitals through the intervening medium, i.e. packing density of the medium. It is known to be 0.9 Å–1 in a fully-packed medium and 2.8 Å–1 in vacuum, and around 1.4 Å–1 in the protein.61-62 The dipole interaction D is expressed as D = D0 / r3, where D0 is a coefficient given by the g-factor of radicals and the angle with respect to the external magnetic field.38 The values of D0 and J0 of P800+MQ– in hRC can be assumed to be comparable to those of P700+PhyQ– in PS I, where β is presumed to be 1.4 Å–1. We calculated the relationship between r and β for P800+MQ– at the |D| = |J| condition (see


Page 12 of 24

Page 13 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Supporting Material and Fig. S5). If r between P800+ and MQ– is the same as that of P700+PhyQ–, then β is estimated to be about 1.2 Å–1, suggesting the more densely-packed protein medium in hRC. Conversely, if β in hRC is 1.4 Å–1 as in PS I, we should assume r to be 21 Å, somewhat shorter than 25 Å between P700 and PhyQ. The long 600-ps electron transfer time from A0– in hRC suggests the distance between MQ and A0, and probably between MQ and P800, to be a bit longer than that between PhyQ and A0,25 supporting the smaller β rather than the shorter r, and also suggesting β to be even lower than 1.2 Å–1. Therefore, we can estimate the location/protein environment of MQ in hRC to be somewhat different from those of PhyQ in PS I.

3.6. Electron transfer in hRC The present ESP-EPR analysis reveals that the P800+MQ– is produced from the P800+A0–. It suggests the sequential electron transfer process of P800→A0→MQ in hRC. On the other hand, the quinone-function in the electron transfer process of hRC is still under debate, and the binding of MQ was not identified in the recent hRC structure.6 We can assume four models based on the results in the present study; Electron is transferred (1) from A0 to MQ directly and then to FX, (2) from A0 to FX directly and then to MQ, (3) from A0 to MQ as well as to FX in parallel and competitively, and (4) MQ does not exist on hRC. The models (1) to (3), but not (4), interpret the observed photoaccumulation of semimenaquinone under continuous illumination.25,31 On the other hand, the model (2) is inconsistent with the result that the P800+MQ– was detected only after the pre-reduction of FX.25 Although the model (3) seems to be better to interpret the observation of the electron transfer to FX after removal of quinone,34 the model (1) is still probable because the slow FX reduction directly by A0 could also be observed in PS I after the extraction of PhyQ.10,63 The fast electron transfer to FX detected even at cryogenic temperature indicates the downhill energy process. It suggests that MQ mediates the electron transfer from A0 to FX in hRC as does PhyQ on the B-branch in PS I, rather than that on the A-branch.64 We, therefore, can interpret the electron transfer mechanism in hRC to be almost



analogous to that in PS I, i.e. MQ should serve as the secondary electron acceptor also in hRC, although further studies are required to clarify the details.

4. Conclusion MQ molecules were not identified in the recently-revealed structure of hRC, although the molecular arrangement showed high similarity to PS I except for the absence of the electron acceptor quinone.6 It suggests either the no function of MQ or loss of intrinsic MQ in hRC during the preparation/crystallization of the hRC from the membranes containing 20-30 MQ per RC. On the other hand, MQ has been identified in the other preparation of purified hRC, which shows a property similar to the hRC preparation used for the crystallization except for the MQ content (see Supporting Material and Figure S1).25-26,29 We detected the photoaccumulation of semimenaquinone as well as the ESP signal in the purified MQ-containing hRC25 so that we assume MQ to be an intrinsic component of hRC. The results in the present study have further confirmed that the P800+MQ– state is derived from the P800+A0– state as fast as to retain clear ESP property, suggesting MQ to be an immediate electron acceptor from A0 in hRC. The spectral simulations indicated that the molecular orientation of MQ resembles that of PhyQ in PS I, although the exchange interaction J is negative and as large as the dipole interaction D in contrast to the P700+PhyQ– state of PS I that shows positive and small J.51-55 The electron transfer system in hRC, thus, was assumed to have the larger exchange interaction factor |J|, i.e. stronger electronic interaction between the cofactors. The discrepancy seems to come from a positional and/or protein environmental deviation of MQ in hRC compared to that of PhyQ in PS I.25 All the RCs seem to be optimized by the fine-tuning of forward and backward electron transfer rates between the electron transfer cofactors.10 In this study, the quinone function in hRC is shown to be somewhat different from that in PS I. The result also shows that the quinone functions, which are well known to be diverse between the type I and II RCs,10-14 are differently tuned even among the type I RCs. Further analysis of the feature of quinone function in hRC that is a primitive RC with the


Page 14 of 24

Page 15 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


homodimeric structure6,9 will give us a clue to clarify the evolutionary scenario of RC structures along with the modification of the quinone function, which has occurred from the homodimeric to the heterodimeric, and from anoxygenic to oxygenic ones.

Associated Content The Supporting Material is available free of charge on the ACS Publications website; (1) HPLC analysis of pigment compositions of cells, RC and highly-purified RC core complexes of Hbt. modesticaldum. (2) Theoretical background of the ESP-EPR signal, (3) Evaluation of the subtraction procedure applied before spectral simulation analysis, and (4) Discussion about positional and protein environmental requirements for MQ in hRC to realize the |D| ≈ |J| condition.

Acknowledgments This work was supported by JSPS Grants-in-Aid for Scientific Research on Innovative Areas No. 17H05724 to H.O. and Grants-in-Aid for Scientific Research (No. 15K07026 to H. O., and Nos. 26440139 and 17K07440 to S. I.) as well as by a JSPS research fellowship for young scientists (No.21008983 to T. K.). We also thank Dr. Hiroyuki Mino at the Department of Physics in Nagoya University for his help with the EPR study, Drs. Masahiro Ishiura at the Center for Gene Research and Takumi Noguchi at the Graduate School of Science, respectively, in Nagoya University for their kind supports during the study, and Dr. Shunsuke Ohashi and Mr. Tomohito Mayumi at the Division of Materials Science in University of Tsukuba for their kind supports during the study.

References (1) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. Structure of the protein subunits in the photosynthetic reaction center of Rhodopseudomonas viridis at 3Å resolution. Nature 1985, 318, 618-624. (2) Niwa, S.; Yu, L.-J.; Takeda, K.; Hirano, Y.; Kawakami, T.; Wang-Otomo, Z.-Y.; Miki, K. Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 angstrom. Nature 2014, 508, 228-232.



(3) Fromme, P.; Jordan, P.; Krauss, N. Structure of photosystem I. Biochim. Biophys. Acta 2001, 1507, 5-31. (4) Amunts, A.; Drory, O.; Nelson, N. The structure of a plant photosystem I supercomplex at 3.4 angstrom resolution. Nature 2007, 447, 58-63. (5) Umena, Y.; Kawakami, K.; Shen, J.-R.; Kamiya, N. Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 angstrom. Nature 2011, 473, 55-U65. (6) Gisriel, C.; Sarrou, I.; Ferlez, B.; Golbeck, J. H.; Redding, K. E.; Fromme, R. Structure of a symmetric photosynthetic reaction center-photosystem. Science 2017, 357, 1021-1025. (7) Olson, J. M.; Blankenship, R. E. Thinking about the evolution of photosynthesis. Photosynth. Res. 2004, 80, 373-386. (8) Vermaas, W. F. J. Evolution of heliobacteria - Implications for photosynthetic reaction center complexes. Photosynth. Res. 1994, 41, 285-294. (9) Sadekar, S.; Raymond, J.; Blankenship, R. E. Conservation of distantly related membrane proteins: Photosynthetic reaction centers share a common structural core. Mol. Biol. Evol. 2006, 23, 2001-2007. (10) Itoh, S.; Iwaki, M.; Ikegami, I. Modification of photosystem I reaction center by the extraction and exchange of chlorophylls and quinones. Biochim. Biophys. Acta 2001, 1507, 115-138. (11) Ohashi, S.; Iemura, T.; Okada, N.; Itoh, S.; Furukawa, H.; Okuda, M.; Ohnishi-Kameyama, M.; Ogawa, T.; Miyashita, H.; Watanabe, T.; Itoh, S.; Oh-oka, H.; Inoue, K.; Kobayashi, M. An overview on chlorophylls and quinones in the photosystem I-type reaction centers. Photosynth. Res. 2010, 104, 305-319. (12) Srinivasan, N.; Golbeck, J. H. Protein–cofactor interactions in bioenergetic complexes: The role of the A1A and A1B phylloquinones in Photosystem I. Biochim. Biophys. Acta 2009, 1787, 1057-1088. (13) Müh, F.; Glöckner, C.; Hellmich, J.; Zouni, A. Light-induced quinone reduction in photosystem II. Biochim. Biophys. Acta 2012, 1817, 44-65. (14) Cardona, T.; Sedoud, A.; Cox, N.; Rutherford, A. W. Charge separation in photosystem II: A comparative and evolutionary overview. Biochim. Biophys. Acta 2012, 1817, 26-43. (15) Oh-oka, H. Type 1 reaction center of photosynthetic heliobacteria. Photochem. Photobiol. 2007, 83, 177-186. (16) Heinnickel, M.; Golbeck, J. H. Heliobacterial photosynthesis. Photosynth. Res. 2007, 92, 35-53. (17) Kobayashi, M.; van de Meent, E. J.; Erkelens, C.; Amesz, J.; Ikegami, I.; Watanabe, T. Bacteriochlorophyll g epimer as a possible reaction center component of heliobacteria. Biochim. Biophys. Acta 1991, 1057, 89-96. (18) Rigby, S. E. J.; Evans, M. C. W.; Heathcote, P. Electron nuclear double resonance (ENDOR) spectroscopy of radicals in photosystem I and related Type 1 photosynthetic reaction centres. Biochim. Biophys. Acta 2001, 1507, 247-259. (19) van de Meent, E. J.; Kobayashi, M.; Erkelens, C.; van Veelen, P. A.; Amesz, J.; Watanabe, T. Identification of 81-hydroxychlorophyll a as a functional reaction center pigment in heliobacteria. Biochim. Biophys. Acta 1991, 1058, 356-362. (20) Mizoguchi, T.; Oh-Oka, H.; Tamiaki, H. Determination of stereochemistry of bacteriochlorophyll gF and 81-hydroxy-chlorophyll aF from Heliobacterium modesticaldum. Photochem. Photobiol. 2005, 81, 666-673. (21) Nitschke, W.; Setif, P.; Liebl, U.; Feiler, U.; Rutherford, A. W. Reaction center photochemistry of


Page 16 of 24

Page 17 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Heliobacterium chlorum. Biochemistry 1990, 29, 11079-11088. (22) Heinnickel, M.; Agalarov, R.; Svensen, N.; Krebs, C.; Golbeck, J. H. Identification of FX in the heliobacterial reaction center as a [4Fe-4S] cluster with an S = 3/2 ground spin state. Biochemistry 2006, 45, 6756-6764. (23) Heinnickel, M.; Shen, G. Z.; Golbeck, J. H. Identification and characterization of PshB, the dicluster ferredoxin that harbors the terminal electron acceptors FA and FB in Heliobacterium modesticaldum. Biochemistry 2007, 46, 2530-2536. (24) Kondo, T.; Matsuoka, M.; Azai, C.; Itoh, S.; Oh-Oka, H. Orientations of iron-sulfur clusters FA and FB in the homodimeric type-I photosynthetic reaction center of Heliobacterium modesticaldum. J. Phys. Chem. B 2016, 120, 4204-4212. (25) Kondo, T.; Itoh, S.; Matsuoka, M.; Azai, C.; Oh-oka, H. Menaquinone as the secondary electron acceptor in the type I homodimeric photosynthetic reaction center of Heliobacterium modesticaldum. J. Phys. Chem. B 2015, 119, 8480-8489. (26) Miyamoto, R.; Iwaki, M.; Mino, H.; Harada, J.; Itoh, S.; Oh-oka, H. ESR signal of the iron-sulfur center FX and its function in the homodimeric reaction center of Heliobacterium modesticaldum. Biochemistry 2006, 45, 6306-6316. (27) Itoh, S.; Iwaki, M.; Ikegami, I. Extraction of vitamin K-1 from Photosystem I particles by treatment with diethyl ether and its effects on the A1 EPR signal and System I photochemistry. Biochim. Biophys. Acta 1987, 893, 508-516. (28) Trost, J. T.; Blankenship, R. E. Isolation of a photoactive photosynthetic reaction center core antenna complex from Heliobacillus mobilis. Biochemistry 1989, 28, 9898-9904. (29) Sarrou, I.; Khan, Z.; Cowgill, J.; Lin, S.; Brune, D.; Romberger, S.; Golbeck, J. H.; Redding, K. E. Purification of the photosynthetic reaction center from Heliobacterium modesticaldum. Photosynth. Res. 2012, 111, 291-302. (30) Redding, K. E.; Sarrou, I.; Rappaport, F.; Santabarbara, S.; Lin, S.; Reifschneider, K. T. Modulation of the fluorescence yield in heliobacterial cells by induction of charge recombination in the photosynthetic reaction center. Photosynth. Res. 2014, 120, 221-235. (31) Muhiuddin, I. P.; Rigby, S. E. J.; Evans, M. C. W.; Amesz, J.; Heathcote, P. ENDOR and special TRIPLE resonance spectroscopy of photoaccumulated semiquinone electron acceptors in the reaction centers of green sulfur bacteria and heliobacteria. Biochemistry 1999, 38, 7159-7167. (32) Miyamoto, R.; Mino, H.; Kondo, T.; Itoh, S.; Oh-Oka, H. An electron spin-polarized signal of the P800+A1(Q)- state in the homodimeric reaction center core complex of Heliobacterium modesticaldum. Biochemistry 2008, 47, 4386-4393. (33) van der Est, A.; Hager-Braun, C.; Leibl, W.; Hauska, G.; Stehlik, D. Transient electron paramagnetic resonance spectroscopy on green-sulfur bacteria and heliobacteria at two microwave frequencies. Biochim. Biophys. Acta 1998, 1409, 87-98. (34) Kleinherenbrink, F. A. M.; Ikegami, I.; Hiraishi, A.; Otte, S. C. M.; Amesz, J. Electron transfer in menaquinone depleted membranes of Heliobacterium chlorum. Biochim. Biophys. Acta 1993, 1142, 69-73. (35) Lin, S.; Chiou, H. C.; Blankenship, R. E. Secondary electron transfer processes in membranes of Heliobacillus mobilis. Biochemistry 1995, 34, 12761-12767.



(36) Kandrashkin, Y. E.; Salikhov, K. M.; van der Est, A.; Stehlik, D. Electron spin polarization in consecutive spin-correlated radical pairs: Application to short-lived and long-lived precursors in type 1 photosynthetic reaction centres. Appl. Magn. Reson. 1998, 15, 417-447. (37) Brettel, K.; Leibl, W.; Liebl, U. Electron transfer in the heliobacterial reaction center: evidence against a quinone-type electron acceptor functioning analogous to A1 in photosystem I. Biochim. Biophys. Acta 1998, 1363, 175-181. (38) Stehlik, D.; Bock, C. H.; Petersen, J. Anisotropic electron-spin polarization of correlated spin pairs in photosynthetic reaction centers. J. Phys. Chem. 1989, 93, 1612-1619. (39) Snyder, S. W.; Rustandi, R. R.; Biggins, J.; Norris, J. R.; Thurnauer, M. C. Direct assignment of vitamin K1 as the secondary acceptor A1 in photosystem I. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 9895-9896. (40) van der Est, A. Light-induced spin polarization in type I photosynthetic reaction centres. Biochim. Biophys. Acta 2001, 1507, 212-225. (41) Nuijs, A. M.; van Dorssen, R. J.; Duysens, L. N. M.; Amesz, J. Excited-states and primary photochemical-reactions in the photosynthetic bacterium Heliobacterium chlorum. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 6865-6868. (42) Kandrashkin, Y. E.; Vollmann, W.; Stehlik, D.; Salikhov, K.; van der Est, A. The magnetic field dependence of the electron spin polarization in consecutive spin correlated radical pairs in type I photosynthetic reaction centres. Mol. Phys. 2002, 100, 1431-1443. (43) Madigan, M. T., The family heliobacteria. In The Prokaryotes (2nd ed.). Balows, A.; Trüper, H. G.; Dworkin, M.; Schleifer, K. H., Eds. Springer, Berlin: 1992; pp 1981-1992. (44) Oh-oka, H.; Kamei, S.; Matsubara, H.; Iwaki, M.; Itoh, S. Two molecules of cytochrome C function as the electron donors to P840 in the reaction-center complex isolated from a green sulfur bacterium, Chlorobium tepidum. FEBS Lett. 1995, 365, 30-34. (45) Ikegami, I.; Katoh, S. Enrichment of photosystem I reaction center chlorophyll from spinach chloroplasts. Biochim. Biophys. Acta 1975, 376, 588-592. (46) Mino, H.; Satoh, J.; Kawamori, A.; Toriyama, K.; Zimmermann, J. L. Matrix ENDOR of tyrosine D+ in oriented photosystem II membranes. Biochim. Biophys. Acta 1993, 1144, 426-433. (47) MacMillan, F.; Hanley, J.; van der Weerd, L.; Knüpling, M.; Un, S.; Rutherford, A. W. Orientation of the phylloquinone electron acceptor anion radical in photosystem I. Biochemistry 1997, 36, 9297-9303. (48) Berthold, T.; Bechtold, M.; Heinen, U.; Link, G.; Poluektov, O.; Utschig, L.; Tang, J.; Thurnauer, M. C.; Kothe, G. Magnetic-field-induced orientation of photosynthetic reaction centers as revealed by time-resolved W-band EPR of spin-correlated radical pairs. J. Phys. Chem. B 1999, 103, 10733-10736. (49) Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C. Structure of the radical pair intermediate in photosystem I by high time resolution multifrequency electron paramagnetic resonance: Analysis of quantum beat oscillations. J. Am. Chem. Soc. 2001, 123, 4211-4222. (50) Hore, P. J. Analysis of polarized EPR spectra. In Advanced EPR: Applications in biology and biochemistry, Hoff, A. J., Ed. Elsevier: Amsterdam, 1989; pp 405-440. (51) Kamlowski, A.; Zech, S. G.; Fromme, P.; Bittl, R.; Lubitz, W.; Witt, H. T.; Stehlik, D. The radical pair state P700+A1- in photosystem I single crystals: Orientation dependence of the transient spin-polarized EPR spectra. J.


Page 18 of 24

Page 19 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Phys. Chem. B 1998, 102, 8266-8277. (52) Zech, S. G.; Hofbauer, W.; Kamlowski, A.; Fromme, P.; Stehlik, D.; Lubitz, W.; Bittl, R. A structural model for the charge separated state P700+A1- in photosystem I from the orientation of the magnetic interaction tensors. J. Phys. Chem. B 2000, 104, 9728-9739. (53) Poluektov, O. G.; Paschenko, S. V.; Utschig, L. M.; Lakshmi, K. V.; Thurnauer, M. C. Bidirectional electron transfer in photosystem I: Direct evidence from high-frequency time-resolved EPR spectroscopy. J. Am. Chem. Soc. 2005, 127, 11910-11911. (54) Berthold, T.; von Gromoff, E. D.; Santabarbara, S.; Stehle, P.; Link, G.; Poluektov, O. G.; Heathcote, P.; Beck, C. F.; Thurnauer, M. C.; Kothe, G. Exploring the electron transfer pathways in photosystem I by high-time-resolution electron paramagnetic resonance: Observation of the B-side radical pair P700+A1B- in whole cells of the deuterated green alga Chlamydomonas reinhardtii at cryogenic temperatures. J. Am. Chem. Soc. 2012, 134, 5563-5576. (55) Santabarbara, S.; Kuprov, I.; Fairclough, W. V.; Purton, S.; Hore, P. J.; Heathcote, P.; Evans, M. C. W. Bidirectional electron transfer in photosystem I: Determination of two distances between P700+ and A1- in spin-correlated radical pairs. Biochemistry 2005, 44, 2119-2128. (56) Goldberg, A. H.; Dougherty, D. A. Effects of through-bond and through-space interactions on singlet triplet energy gaps in localized biradicals. J. Am. Chem. Soc. 1983, 105, 284-290. (57) Kobori, Y.; Sekiguchi, S.; Akiyama, K.; Tero-Kubota, S. Chemically induced dynamic electron polarization study on the mechanism of exchange interaction in radical ion pairs generated by photoinduced electron transfer reactions. J. Phys. Chem. A 1999, 103, 5416-5424. (58) Calvo, R.; Abresch, E. C.; Bittl, R.; Feher, G.; Hofbauer, W.; Isaacson, R. A.; Lubitz, W.; Okamura, M. Y.; Paddock, M. L. EPR study of the molecular and electronic structure of the semiquinone biradical QA-QB- in photosynthetic reaction centers from Rhodobacter sphaeroides. J. Am. Chem. Soc. 2000, 122, 7327-7341. (59) Kobori, Y.; Yago, T.; Akiyama, K.; Tero-Kubota, S.; Sato, H.; Hirata, F.; Norris, J. R. Superexchange electron tunneling mediated by solvent molecules: Pulsed electron paramagnetic resonance study on electronic coupling in solvent-separated radical ion pairs. J. Phys. Chem. B 2004, 108, 10226-10240. (60) Coffman, R.; Buettner, G. A limit function for long-range ferromagnetic and antiferromagnetic superexchange. J. Phys. Chem. 1979, 83, 2387-2392. (61) Moser, C. C.; Keske, J. M.; Warncke, K.; Farid, R. S.; Dutton, P. L. Nature of biological electron transfer. Nature 1992, 355, 796-802. (62) Page, C. C.; Moser, C. C.; Chen, X.; Dutton, P. L. Natural engineering principles of electron tunnelling in biological oxidation–reduction. Nature 1999, 402, 47-52. (63) Itoh, S.; Iwaki, M. Vitamin K1 (phylloquinone) restores the turnover of FeS centers in the ether-extracted spinach PS I particles. FEBS Lett. 1989, 243, 47-52. (64) Santabarbara, S.; Reifschneider, K.; Jasaitis, A.; Gu, F.; Agostini, G.; Carbonera, D.; Rappaport, F.; Redding, K. E. Interquinone electron transfer in photosystem I as evidenced by altering the hydrogen bond strength to the phylloquinone(s). J. Phys. Chem. B 2010, 114, 9300-9312.



Figure 1. Angular dependence of the ESP-EPR spectrum of P700+PhyQ– (A) and P700+FX– (B) in PS I and P800+MQ– (C) and P800+FX– (D) in hRC. Upper spectrum in each panel was measured in the random-oriented membranes (A, B, and D) and hRCc (C). The middle and bottom were measured in the oriented membranes placed at an angle of 0o and 90o, respectively, between the membrane normal and the external magnetic field. The ESP intensities at 10 µs after flash excitations were plotted. Solid lines (black) in panel A indicate simulated spectra. Experimental conditions: microwave power, 1 mW; microwave frequency, 9.527 GHz; modulation amplitude, 0.4 mT at 100 kHz; time constant, 0.01 ms; temperature, 14 (A), 250 (B), and 10 K (C and D).


Page 20 of 24

Page 21 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Spectral simulation for the P800+MQ– ESP state, calculated from the singlet term with D = –0.17 mT and different J values in the random-oriented hRCc (A) and oriented membrane (B and C). Upper panels show the experimental spectra obtained from Figure 1, where the dotted (red) and broken (blue) lines indicate the P800+MQ– and P800+FX– spectra, respectively, after normalization. Solid line (green) indicates the difference spectrum estimated by subtracting the P800+FX– spectrum from the P800+MQ– spectrum. The amplitudes of spectra in C were normalized with respect to those in B. The simulation in the lower panels were performed with the same quinone-orientation as that of PhyQ in PS I (gx // Zd).



Figure 3. Spectral simulation of angular dependent ESP-EPR spectra of P800+MQ– state with different orientation of MQ. The spectra in (A) represent the spectra of P800+MQ– after subtracting normalized P800+FX– signals, shown as “difference spectrum” in Figure 2, upper panel. In B-D, simulated spectra were calculated from the singlet term with D = –0.17 mT, J = –0.2 mT, and the arrangement of MQ at gx // Zd (B), gy // Zd (C), and gz // Zd (D). Each panel shows the spectra in the random-orientation (top) and at 0o (middle) and 90o (bottom) with respect to the external magnetic field.


Page 22 of 24

Page 23 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. ESP patterns observed in the present study, and properties of radical pairs contributing to the ESP pattern.



Table of Contents (TOC) Image


Page 24 of 24

Light-Induced Electron Spin-Polarized (ESP) EPR Signal of the P800+

Recommend Documents