Location of the High-Affinity Mn2+ Site in Photosystem II

Article pubs.acs.org/JPCB

Location of the High-Affinity Mn2+ Site in Photosystem II Detected by PELDOR Mizue Asada and Hiroyuki Mino* Division of Material Science (Physics), Graduate School of Science, Nagoya University, Furo-cho, Chikusa, Nagoya, 464-8602, Japan ABSTRACT: The location of the high-affinity Mn2+ site in apo-photosystem (PS) II was investigated by pulsed EPR. The electron−electron magnetic dipole interaction of 1.7 MHz between the YD• radical and Mn2+ ion was observed using the pulsed electron− electron double resonance (PELDOR) technique, and the Mn2+ ion was bound to one apo-PS II in the absence and presence of Ca2+. PELDOR signals were calculated using the previously determined spin distribution on the YD• radical and its known position in the crystal structure, assuming that the specific Mn2+ site was located in the oxygen evolving complex. The results show that the high-affinity Mn2+ site is located at the position denoted by Mn4(A) in the native crystal structure. The Mn2+ is coordinated with axial ligands Asp170 and Glu333 in the D1 polypeptide.

1. INTRODUCTION The earth is a unique planet with vibrant flora and fauna. Over the years, the earth’s atmosphere has been transformed considerably owing to photosynthetic oxygen evolution. The explanation of the oxygen evolution mechanism is essential for understanding the earth’s history. Oxygen evolution occurs in the Mn cluster of photosystem (PS) II, which is a transmembrane protein complex consisting of D1/D2 subunits with pseudo C2 symmetry, surrounded by proteins. The detailed molecular structure of PS II has been studied for years,1−3 with the crystal structure being reported with 1.9 Å resolution X-ray crystallography,4 and more recently using X-ray free electron laser (XFEL).5 These excellent advances in structural information facilitated clearer analysis of the oxygen evolving complex (OEC) structure. However, the mechanism of oxygen evolution is still unclear. Oxygen evolution is catalyzed by the Mn cluster in PS II, which consists of four Mn, one Ca, and five oxygen atoms, and is surrounded by amino acids (Figure 1). Four Mn atoms, labeled Mn1−4, and one Ca atom are bridged with oxygen atoms in a distorted chair structure. The amino acid residues coordinated to the Mn atoms are D1-H332, D1-D342, and D1E189 for Mn1, D1-D342, D1-A344, and CP43-E354 for Mn2, D1-E333 and CP-43-E354 for Mn3, and D1-D170 and D1E333 for Mn4. The Mn cluster is oxidized by sequential electron transfer via YZ by light-induced charge separation in P680 bound to D1/D2 proteins (see reviews6,7). YD residue is located in the D2 protein with pseudo C2 symmetry in relation to YZ in the D1 protein, and keeps a stable radical form. In higher plants, extrinsic subunits PsbO, PsbP, and PsbQ are associated with the lumenal side of the PS II complex (see review8). These extrinsic subunits are considered to maintain the inorganic cofactors Ca2+ and Cl− in the oxygen evolving complex, and thus help optimize the water oxidizing activity. Photoactivation is the process of Mn-cluster reconstitution by light-induced reactions. Since the early reports,9−12 many studies and models of the process have been proposed (see © 2015 American Chemical Society

Figure 1. Structure of the oxygen-evolving complex of PS II and its coordinating environment based on X-ray crystallography (PDB: 4UB6).

reviews7,13−15). The two-quantum mechanism is one of the proposed photoactivation models.11,16 In this model, the reaction is initiated by mixing NH2OH-treated apo-PS II with Mn2+ and Ca2+ ions. The first step is incorporation of one Mn2+ ion onto a high-affinity site of dark-adapted apo-PS II (denoted as the IM0 state15). After illumination, Mn2+ is oxidized (IM1 state). Through structural rearrangement in the dark state, Ca2+ Received: April 27, 2015 Revised: July 3, 2015 Published: July 23, 2015 10139

DOI: 10.1021/acs.jpcb.5b03994 J. Phys. Chem. B 2015, 119, 10139−10144

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The Journal of Physical Chemistry B binds near Mn3+ in apo-PS II (IM1*),17 indicating that Ca2+ binding causes the rearrangement of OEC and induces the subsequent photoactivation steps. Further illumination results in the formation of the first stable intermediate Mn3+−Mn3+ (IM2 state), which is monitored by XAS.18 There is one specific high-affinity Mn2+ site in the initial state of photoactivation that is oxidized to Mn3+ by illumination.19 Site-directed mutagenesis has shown that Asp170 is associated with the initial assembly of the Mn cluster.20 However, there is no direct evidence about the exact location of Mn2+. Campbell et al. observed an EPR signal corresponding to the photooxidized Mn3+ ions bound to apo-PS II; however, no such signal was observed in the D1-Asp170Glu mutant.21 On the other hand, the site-directed mutant D1-Asp170His was found to be active.22 Pulsed electron−electron double resonance (PELDOR) is a powerful method that helps detect the magnetic dipole interaction between two electron spins by deriving the relative position between the electrons.23 The stable YD• radical has so far been a benchmark in determining the relative position of the redox components in PS II. The high-resolution crystal structures provide new insight into the magnetic structure deduced by EPR measurements. Recently, we have performed PELDOR experiments to determine the spin distribution on the Mn cluster, by utilizing the coordinates of the Mn cluster and tyrosine radical YD• in the crystal structure.24 In this paper, we report the location of the high-affinity Mn2+ site in apo-PS II, deduced by the PELDOR technique. The obtained results indicate that the high-affinity Mn2+ site is located at the immobile position close to the native OEC structure. By relating with the crystal structure, details of the exact position of the high-affinity site are discussed.

2.3. PELDOR Simulations. The PELDOR spectra were simulated as described previously24 with few modifications. The PELDOR signal amplitude X(τ′) as a function of the time interval τ′ between the first and second pulses was taken as28,29 X(τ′) ∝ 1 − p[1 − cos(2πDτ′)]

(1)

with D=

∑ ρi i

g1g2β 2 hR i 3

(1 − 3 cos2 Θi)

(2)

where ρi is the spin density at the ith (i = 1−7) carbon/oxygen atom of the YD• molecule, as designated earlier.24,30 Ri is the length between the ith (i = 1−7) carbon/oxygen atom of the YD• and the Mn2+ atom. Θi is the angle between the external magnetic field H and the distance vector Ri. Parameters g1 and g2 are the g-factors for two electron spins, assumed to be 2.0046 for YD• and 2.0093 for Mn2+. The signal amplitude I(τ′) is given by the surface integration: I(τ′) =

∬ X(τ′) sin θ dθ dφ

(3)

3. RESULTS Figure 2 shows the CW-EPR spectra of a buffer containing MnCl2 (black lines) and the supernatant after centrifugation

2. MATERIALS AND METHODS 2.1. Sample Preparation. PS II membranes were prepared as described.25,26 The membranes were suspended in a buffer containing 400 mM sucrose, 20 mM Mes/NaOH, and 2 M NaCl (pH 6.5) at 1 mg of Chl/mL and were incubated for 20 min at 4 °C. Thereafter, the membranes were washed with a buffer containing 400 mM sucrose, 20 mM NaCl, 1 mM NH2OH, and 20 mM Mes/NaOH (pH 6.5), and centrifuged at 35 000 × g for 20 min.19 After incubating for 1 min at 4 °C, 0.5 mM Na•EDTA was added. The samples were washed 3 times with a buffer containing 400 mM sucrose, 20 mM NaCl, and 40 mM Mes/NaOH (pH 6.5). The membranes were illuminated for 1 min at 4 °C with room light to ensure YD• oxidation. In the case of the presence of Ca2+ ion, 10 mM CaCl2 was added to the buffer. After centrifuging at 35 000 × g for 20 min, the precipitate was transferred into EPR tubes. The amount of Mn2+ bound to apo-PS II in the supernatant was quantified. 2.2. EPR Measurements. CW-EPR measurements were carried out by a Bruker ESP-300E EPR spectrometer with a standard cavity (TE102). The amount of Mn2+ in the solution was measured by comparing with MnCl2 solution contained in 50 μL capillary tubes at room temperature. Pulsed EPR and ELDOR were carried out by Bruker E580 with an Oxford Instruments liquid helium cryostat (CF935P, oxford GB). A π/ 2-τ-π sequence was used to measure the ESE field swept spectra. The time interval τ between the microwave pulses was set to 200 ns. A π/2-τ1-π-τ1-τ2-π sequence with a time interval τ1 of 200 ns and τ2 of 1400 ns was used for four-pulsed ELDOR.24,27 A microwave synthesizer (HP83751A, HewlettPackard) was utilized as the second microwave source.24

Figure 2. Field swept CW-EPR spectra of Mn2+ in the buffer with 20 μM MnCl2 (black lines) and the supernatant after centrifuging the mixture of the buffer and apo-PS II at 1 mg of Chl/mL (red lines) in the absence (a) and in the presence (b) of Ca2+ observed at room temperature. Experimental conditions: microwave frequency, 9.79 GHz; microwave power, 64 mW; field modulation amplitude, 8 G at 100 kHz; time constant, 3 ms.

(red lines) of PS II membranes in (a) the absence and (b) the presence of Ca2+ at room temperature. Apo-PS II membranes of 1 mg of Chl/mL treated with NH2OH were suspended in 20 μM MnCl2 and centrifuged. The observed six line signals are attributed to the free Mn2+ ions. The decrease in the amplitude of these signals post centrifugation indicates that the Mn2+ bound to apo-PS II precipitated out, and that nonbound Mn2+ remained in the supernatant.19 After analyzing the data, the numbers of Mn2+ ions bound to apo-PS II were estimated to be 1.4 per PS II in the absence of Ca2+ and 0.4 per PS II in the presence of Ca2+, respectively. The amount of Mn2+ bound to apo-PS II in the presence of Ca2+ was less than when Ca2+ was absent. The results confirm that Ca2+ competes with Mn2+ for both Mn2+-specific and nonspecific sites, thereby regulating Mn2+ binding to the high-affinity site.11,12,31 10140

DOI: 10.1021/acs.jpcb.5b03994 J. Phys. Chem. B 2015, 119, 10139−10144

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Figure 4A shows the PELDOR signals of the apo-PS II after NH2OH treatment, centrifugation, and washing with (a) 20 μM Mn2+-containing buffer in the absence of Ca2+, with (b) 20 μM Mn2+ and with (c) 40 μM Mn2+-containing buffer in the presence of Ca2+. The Mn2+ signals were observed at 344 mT (blank arrow, Figure 3) and pumped on the YD• signal at 338 mT (filled arrow, Figure 3). To reduce unwanted ESEEM beats,27,32 eight different signals with eight τ2 lengths (τ2 = 1400−1456 ns with Δτ2 = 8 ns) were averaged. Panel B shows the signals obtained after subtracting the linear baseline from panel A. The PELDOR signal in the absence of Ca2+ shows the first negative peak at τ′ = 296 ns and the first positive peak at τ′ = 584 ns (Figure 4B(a)). The second negative peak around τ′ = 960 ns for (a) 20 μM Mn2+ in the absence of Ca2+ and for (c) 40 μM Mn2+ in the presence of Ca2+ was neglected, because the peak could result from baseline drift. Assuming the point dipole approximation, the distance between YD• and the Mn2+ sites was estimated to be 30.5 Å. Clear oscillation of the PELDOR signals implies that the Mn2+-binding site is located in the immobile site with a narrow distance distribution, evaluated to be 1.6 Å using the Deeranalysis 2011 program.33 In the presence of Ca2+, the PELDOR signal had the same period but the modulation depth was shallower than that in the absence of Ca2+ (traces a and b in Figure 4B). The frequency of the PELDOR signal at high concentrations of Mn2+ was the same as that at low concentrations of Mn2+ (traces b and c), indicating that the single high-affinity sites of Mn2+ are being detected in the observed range. The PELDOR modulation depth for high concentrations was deeper than that of low concentrations (traces b and c), which is consistent with the fact that Ca2+ ions and Mn2+ ions compete to bind to the highaffinity site.12,34,35

Figure 3 shows the ESE-field swept spectra of the postcentrifugation pellet samples, washed in (a) the absence

Figure 3. ESE field swept spectra of the apo-PS II with MnCl2 in the absence (black lines, a and d) and in the presence (red lines, b and c) of CaCl2 before illumination (bold lines, a and b) and after illumination (dotted lines, c and d). The samples were illuminated for 3 min at 273 K and were frozen in a liquid N2 bath immediately (within 10 s). The signal amplitudes were normalized by the intensity of YD• at 344 mT. Experimental conditions: microwave frequency, 9.49 GHz; pulse length, 16 ns for π/2 pulses and 24 ns for π pulses; interval tau between the mw pulses, 200 ns; repetition time, 100 μs; temperature, 8 K. The arrows show the resonant magnetic fields for the observation (blank) and pumping (filled) microwave frequencies in the PELDOR measurement.

and (b) the presence of 10 mM Ca2+ ions in the dark state. The signal intensities were normalized with respect to the YD• signal intensity at g = 2. The broad signals with 60 mT width centered at g = 2 arise from Mn2+ bound to PS II. The signal in the presence of Ca2+ is smaller than that in the absence of Ca2+, which is consistent with the number of Mn2+ ions estimated from CW-EPR at room temperature (Figure 2). The dotted lines show the spectra after 3 min of illumination at 273 K in the absence and presence of Ca2+. The Mn2+ signal in the absence of Ca2+ disappeared after illumination, indicating that Mn2+ binds to the high-affinity site during multiturnovers.

4. DISCUSSION In this work, we have reported the PELDOR oscillation caused by the interactions between YD• and Mn2+ bound to apo-PS II. Assuming the point dipole approximation, the distance between YD• and the Mn2+ site is estimated to be 30.5 Å, which is close to the location of the Mn cluster in native OEC. The distances

Figure 4. (A) Pulsed ELDOR signals of the interactions between YD• and Mn2+. The signals were obtained in (a, b) 20 μM MnCl2 and (c) 40 μM MnCl2 in (a) the absence of Ca2+ and in (b, c) the presence of Ca2+. (B) The signals after subtracting linear decay from the panel A signals. Experimental conditions: observation mw frequency, 9.49 GHz; pumping mw pulse frequency, 9.68 GHz; pulse length, 16 and 24 ns; time interval between first and third pulses τ2, with a summation of eight time length in the range τ2 = 1400−1456 ns; magnetic field, 350 mT; repetition time, 250 μs; temperature, 8 K. 10141

DOI: 10.1021/acs.jpcb.5b03994 J. Phys. Chem. B 2015, 119, 10139−10144

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and 30.7 Å,4,5 respectively. In order to obtain the accurate position of the high-affinity site of Mn2+, the spin density distribution on YD• must be included.24 Initially, we assumed that the Mn2+ affinity site is located within the metal ion cluster in OEC of the native PS II and accordingly calculated the PELDOR oscillations. Figure 5 shows the experimental (a) and simulated PELDOR signals (b−f). The first positive peaks are τ′ = (b) 526 ns for Mn1, (c) 580 ns for Mn4, (d) 610 ns for Ca, (e) 638 ns for Mn3, and (f) 686 ns for Mn2, respectively. The Mn4 location is in good agreement with the experimental results. As PELDOR oscillation indicates that the distance distribution is relatively narrow, the possibility of monodentate coordination of Mn2+ with the single carboxylate should be excluded because of the distance distribution caused by the large rotation of the amino acid residue. As Mn4 is the only Mn atom axially bound to the amino acids in the native structure, Mn2+ can easily bind to the native position between Asp170 and Glu333 even under depleted OEC conditions. The removal of the Mn cluster might cause structural changes around the cofactors. The positions determined by PELDOR are located on the sphere-like curved surface centered on the YD• molecule. Figure 6 illustrates possible locations of the high-affinity Mn2+ site in the crystal structure (PDB 4UB6), depicted as the gray surface, where the points

Figure 5. PELDOR signals of (a) experimental and (b−f) simulated signals assuming that Mn2+ is located on one of the cofactor sites in OEC. Trace a is the same as that in Figure 4B(a). The simulated traces are for (b) Mn1, (c) Mn4, (d) Ca, (e) Mn3, and (f) Mn2 in the crystal structure depicted in Figure 1.

between the aromatic ring center of YD• and the cofactors of OEC, Mn1, Mn2, Mn3, Mn4, and Ca are 29.3, 32.0, 31.2, 30.2,

Figure 6. Stereo view of the probable position of the Mn2+ high-affinity site. Curved surfaces (dots) represent the coordinates corresponding to the PELDOR simulation. The positions where PELDOR signals have the first negative peak with τ′ = 290−320 ns and the positive peak at τ′ = 575−595 ns were overlaid on the crystal structure (PDB: 4UB6). Panels A and B show the view from different orientations. 10142

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The Journal of Physical Chemistry B with the first negative PELDOR peaks at τ′ = 290−320 ns and the first positive PELDOR peaks at τ′ = 575−595 ns are shown. Mn4 is located on the curved surface. There are two possible Mn2+ sites which have bidentate coordination with two ligand CO in opposite sides. One is the Mn4 site between Asp170 and Glu333, and the other is the site between Glu189 and Ala344. Although the position between Glu189 and Ala344 is not the position for any Mn or Ca atoms in the native crystal structure and the distance between the amino acids is slightly long, Mn2+ might bind to these amino acids if there is a structural modification. However, this is not consistent with the results of the site-directed mutant.20,21 When the high-affinity Mn2+ site is located between Asp170 and Glu333, the Mn2+-connecting axis is determined by the CO orientation in the carboxylate of Asp170 and Glu333, which is in the same as Mn4 in the native Mn cluster. Mn2+ binding between Asp170 and Glu333 would mold a partial prototype of the Mn cluster. Through the photoactivation process, the Mn2+ ion at the high-affinity site is oxidized by illumination. The site-directed mutagenesis results showed that Asp170Glu does not inhibit formation of the OEC, but the residue is not a direct ligand of the Mn3+ ion formed by lightinduced oxidation of Mn2+.20,21 It would be caused by the change of the axial binding length to the Mn ion, which the axial binding length of Mn3+ is longer than that of Mn2+. According to the photoactivation model, the rearrangement from the intermediate state IM1 to the IM1* requires a long time (∼70 s) and has a low efficiency.36 Axial binding of Mn3+ of IM1 might be hindered by the shortened length between Asp170Glu and Glu333, resulting in the weakness of the affinity of Mn3+ in the site. This molecular mechanism would explain the reason why Asp170Glu maintains high O2 activity,20 where release of Mn3+ ion is essential for photoactivation. In conclusion, we successfully determined the location of the high-affinity Mn2+ site in apo-photosystem II. The Mn2+ ion is located at the position occupied by Mn4 in native OEC, indicating that Mn2+ is coordinating to Asp170 and Glu333. The current results along with the new crystal structure help provide yet another clue about the mechanism of the photoactivation process.

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cyanobacteria) of the D2 subunit in PS II; YZ, Tyr161 of the D1 subunit in PS II

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-52-7892882. Fax: +81-52-789-2882. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Program for Leading Graduate Schools “Integrative Graduate Education and Research in Green Natural Sciences”, MEXT, Japan, and a MEXT/JSPS Grant-in-Aid for Exploratory Research No. 26620003 to H.M., and for JSPS Fellows No. 15J11091 to M.A.

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ABBREVIATIONS EPR, electron paramagnetic resonance; PELDOR, pulsed electron−electron double resonance; ESE, electron spin echo; CW, continuous wave; MW, microwave; Mes, 2-morpholinoethasulfonic acid; PS II, photosystem II; OEC, oxygen-evolving complex; Y D , Tyr161 for higher plants (Tyr160 for 10143

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