Calcium, Strontium, and Protein Dynamics during the S2 to S3

Sep 23, 2013 - that strontium substitution has a dramatic effect on the infrared spectrum of the S2 to S3 transition, reducing the intensity of all sp...
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Calcium, Strontium, and Protein Dynamics during the S2 to S3 Transition in the Photosynthetic Oxygen-Evolving Cycle Brandon C. Polander and Bridgette A. Barry* Department of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia 30332, United States S Supporting Information *

ABSTRACT: Photosystem II (PSII) catalyzes the oxidation of water at a Mn4CaO5 cluster. The mechanism of water oxidation requires four sequential photooxidation events and cycles the OEC through the S0−4 states. Oxygen is released during a thermal transition from S4 to S0, and S1 is the dark stable state. Calcium is required for activity, and, of substituted cations, only strontium supports activity but at a lower steady-state rate. The S1 to S2 transition corresponds to a Mn oxidation reaction. Previously, we used divalent ion substitution to provide evidence that calcium activates water and that an internal water cluster (W5+) is protonated during the S1 to S2 transition. For the next transition, S2 to S3, either a Mn or a ligand oxidation event has been proposed. Here, we use strontium reconstitution and reaction-induced FT-IR spectroscopy to study this transition. We show that strontium substitution has a dramatic effect on the infrared spectrum of the S2 to S3 transition, reducing the intensity of all spectral bands in the mid-infrared region (1600− 1200 cm−1). However, the S3 to S0 and S0 to S1 spectra and the flash dependence of W5+ decay are not significantly altered in strontium PSII. The observed decrease in mid-infrared intensity is consistent with inhibition of a protein reorganization event, which may be associated with a strontium-induced change in S3 charge distribution. These data provide evidence that strontium replacement alters the S2 to S3 conformational landscape. SECTION: Biophysical Chemistry and Biomolecules

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(Figure 1A)7 and Sr PSII (Figure 1B)17 does reveal small (0.1− 0.2 Å) changes in the distance to ordered water molecules and carboxylate ligands (Figure 1A and B). While it is not known which S state is represented in the Ca PSII and Sr PSII crystals,18,19 these structures provide important starting points in understanding the PSII mechanism. As probed by X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS), the S1 to S2 transition corresponds to a Mn oxidation reaction and does not involve significant rearrangement of the metal cluster.7,17,20−24 On the other hand, the S2 to S3 transition does cause significant change to metal-to-metal distances in the Mn4CaO5 cluster.22−25 A Mn oxidation event on this transition was supported by XANES, conducted at room temperature.22 However, other XANES and EXAFS studies supported the conclusion that an oxygen ligand is oxidized during this transition. When Sr is substituted for Ca, altered electron paramagnetic (EPR) signals from the S2 state have been observed.26−28 Specifically, an alteration in hyperfine couplings in the g = 2.0, S = 1/2 multiline signal was detected. Also, an increased intensity in an S = 5/2, g = 4.1 signal was observed (see ref 29 for an example). Interestingly, electron nuclear double resonance

n oxygenic photosynthesis, photosystem II (PSII) carries out light-driven water oxidation.1−5 PSII is a multisubunit transmembrane protein complex, which is composed of at least 20 subunits (17 membrane spanning, 3 extrinsic).6,7 The major intrinsic subunits, which bind the redox-active cofactors, are D1, D2, CP43, and CP47.8 The roles and stoichiometries of extrinsic subunits, such as PsbO, PsbP, and PsbQ, and other intrinsic subunits, such as cytochrome b559, have been discussed.9−12 The oxygen-evolving complex (OEC), a Mn4CaO5 cluster (Figure 1A), cycles through five distinct states (S0−S4).13 These S states (Figure 2A) represent the storage of oxidizing equivalents at the OEC. S1 is the darkstable state, and water is oxidized after the accumulation of four oxidizing equivalents (S3 to S0 transition).14 Calcium is an essential component of the OEC,15,16 but the specific role of calcium in water oxidation has not yet been defined. It has been shown that removal of the extrinsic polypeptides, PsbP and PsbQ, results in the loss of OEC calcium (for review, see ref 2). Calcium prevents reduction of the OEC by some exogenous reductants.3 Other ions may occupy the calcium-binding site in the OEC (Figure 1A), but only strontium is capable of supporting oxygen evolution, although at a decreased steady-state rate.3 The recent 2.1 Å crystal structure of strontium-substituted PSII (Sr PSII, Figure 1B) has concluded that there are no large alterations in the geometry of the Mn ions when compared to Ca PSII.17 However, comparison of the crystal structures of Ca PSII © 2013 American Chemical Society

Received: July 29, 2013 Accepted: September 16, 2013 Published: September 23, 2013 3356

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water ligands (Ca (11.41), Sr (12.67)).3 Recently, we investigated the role of calcium in water activation by means of Fourier transform infrared (FT-IR) spectroscopy in the 3200−1200 cm−1 region.33−35 For a previous study at lower wavenumber, see ref 36. In this technique, laser flashes are used to advance through the S state cycle, and difference spectra are recorded for each transition (see early examples, refs 37 and 38). In the S1 to S2 transition, we detected a high wavenumber band from a protonated water cluster, W5+ (Figure 2A), and we

Figure 1. Structures of Ca PSII (A) and Sr PSII (B) at 1.9 and 2.1 Å resolution, respectively.7,17 PSII was isolated from the cyanobacterium T. vulcanus. Colors: oxygen from water, blue; bridging oxygen ligand, red; calcium, yellow; strontium, magenta. Amino acids are shown as sticks, with carbon green and oxygen red. Distances to assigned ligands are shown as dashed lines, with distances measured in angstroms. Only two of seven ligating amino acids are shown.

(ENDOR) experiments suggested that the electronic coupling of the cluster is not altered significantly in the S2 state of Sr PSII, as compared to the S2 state in Ca PSII. The observed g = 2.0 and 4.1 changes were recently attributed to a small variation in distance between a bridging oxygen ligand and Mn ion.5 While the structure of the S2 state is not expected to be sensitive to Ca/Sr substitution, some evidence suggests that the S3 state is altered by metal substitution. Ca removal inhibits this transition.30 Also, in Sr XANES and EXAFS,23 the S3 data were consistent with elongated Mn−Mn distances and shortened Mn−Sr distances compared to the S1 and S2 states. By contrast in Ca PSII, EXAFS revealed increases in Mn−Mn and Mn−Ca distances.22,24,25 Several different experimental approaches have suggested that calcium serves to bind a water molecule and to activate substrate water.28,31−33 The functional properties of strontium have been attributed to the ionic radii of calcium (1.12 Å) and strontium (1.26 Å) and the similarity of the predicted pKa for

Figure 2. Structural rearrangement and proton-transfer reactions preceding water oxidation in PSII. (A) The S state cycle of PSII, showing a mechanism of electron and proton release from the OEC. A proton is transferred to an intermediary water cluster (W5) during the S1 to S2 transition and is then released from PSII on later transitions. (B) The generation of a Ca-minus-Sr double difference spectrum by subtraction of a Sr PSII (top right, blue) FT-IR difference spectrum from a Ca PSII (top left, red) spectrum.

showed that the observation of this band during the S1 to S2 transition depended on the presence of calcium and was inhibited by addition of ammonia.35 In addition, during the S1 to S2 transition, peptide carbonyl frequencies indicated that the observed changes in hydrogen bonding were calcium-dependent. This work suggested that calcium regulates hydrogen bonding in the OEC and that calcium activates water during the S1 to S2 transition.33,34 3357

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time scale employed, this approach monitors long-lived structural changes in the OEC.29,41 It has been reported that these spectra are dominated by donor side dynamics and oscillate with period four.37,38 Spectra were normalized for the protein concentration, path length, and charge separation (see the description in refs 33−35). A one-to-one subtraction of these corrected FT-IR data gives a Ca-minus-Sr double difference spectrum, associated with each flash (Figure 2B). To appear in this double difference spectrum, bands must be changed in amplitude or frequency by the transition and must also respond to the replacement of calcium with strontium. Figure 3 presents the 1900−1200 cm−1 region of the reaction-induced FT-IR spectrum for each accessible S state (S0−S3) (Figure 3A−D), corresponding to S2-minus-S1, one flash (Figure 3A); S3-minus-S2, two flashes (Figure 3B); S0minus-S3, three flashes (Figure 3C); and S1-minus-S0, four flashes (Figure 3D). These spectra were recorded either from Ca PSII (red) or Sr PSII (black). For comparison to the S2 to S3 data (discussed below), Figure 3A presents the results on the first flash, which have been described previously and attributed to hydrogen-bonding changes in the peptide carbonyl−water network.34 For the S1 to S2 transition (first flash, Figure 3A), shifts of amide I (CO stretch, 1650 cm−1) bands are observed when Sr is substituted for Ca. These changes are accompanied by alterations in the amide II (CN stretch/NH bend, 1550 cm−1) and amide III (NH bend/CN stretch 1400− 1250 cm−1) regions.42 These spectral changes are clearly distinguished in the double difference spectra (Ca PSII-minusSr PSII) (Figure 4), when compared to a negative control (Figure 4E). The observed band shapes have been attributed to frequency shifts due to an alteration in hydrogen bonding. This

In this current study, we provide information concerning the protein structural dynamics occurring during the S2 to S3 transition. PSII samples were isolated,33 calcium was depleted, and then either calcium or strontium was reconstituted by a method that we have described previously (see refs 33 and 35 and the Supporting Information). The activities of the reconstituted preparations were 1240 ± 20 μmol O2 (mg chl· h)−1 for Ca PSII and 1020 ± 30 μmol O2 (mg chl·h)−1 for Sr PSII, similar to that of an untreated control (1310 ± 30 μmol O2 (mg chl·h)−1) at the same pH (pH 7.5). Reaction-induced FT-IR spectroscopy was used to monitor the PSII structural dynamics, induced by a series of laser flashes. These spectra reflect frequency and intensity shifts in amide vibrational bands (amides I, II, and III) and, possibly, in amino acid side chains, which provide ligation or undergo a conformational change.39 These spectra will reflect any charge redistribution events in the metal cluster, such as an oxidation reaction, because ligand frequencies and amplitudes are sensitive to the metal oxidation state.40 To obtain the data presented here, FT-IR spectra were monitored on the 15 s time scale after illumination of these samples with short (10 ns), actinic laser flashes (532 nm). Procedures to preserve the integrity of the samples were used, as described in the Supporting Information and our previous work.33−35 Difference FT-IR spectra, associated with each S state transition, were constructed (Figure 3). For example, in the S3-minus-S2 spectrum, unique bands of the S3 state are positive, and unique bands of the S2 state are negative. On the

Figure 3. Mid-infrared region of flash-induced, reaction-induced FTIR spectra at 263 K. Spectra were acquired from Ca PSII (red) or Sr PSII (black). (A) S2-minus-S1 spectrum, (B) S3-minus-S2 spectrum, (C) S0-minus-S3 spectrum, and (D) S1-minus-S0 spectrum. In (E), a baseline collected prior to the first flash is shown. Y-axis tick marks represent 1 × 10−4 absorbance units. Spectra are averages of data from 31 (A−D, red) and 16 (A−E, black) samples.

Figure 4. Ca-minus-Sr double difference spectra, constructed from the data presented in Figure 3. In (B), bands shaded in blue are assigned to Sr PSII; bands shaded in red are assigned to Ca PSII. A control double difference (E) spectrum was generated by subtraction of onehalf of the data in Figure 3A, black, from the other and division by √2. Y-axis tick marks represent 1 × 10−4 absorbance units. 3358

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Figure 3B presents the reaction-induced FT-IR spectrum acquired upon a second actinic flash in Ca PSII (red) and Sr PSII (black). Interestingly, the S3-minus-S2 difference spectrum of Sr PSII (Figure 3B, black) exhibited marked changes in band position and frequency compared to Ca PSII (Figure 3B, red), and these data are also distinct compared to those of the first flash (Figure 3A). For example, the Sr spectrum lacks negative bands at 1569 and 1548 cm−1 and positive bands at 1530 and 1509 cm−1. In Sr PSII, reduced intensity is also observed at +1445 and −1399 cm−1 and in the +1340−1220 cm−1 region. A band shift of +1693 (Ca PSII) to 1657 (Sr PSII) cm−1 is evident. These alterations are all observed in the Ca-minus-Sr spectrum (Figure 4B), where the bands are significant relative to the negative control (Figure 4E). To summarize, the Sr PSII S3-minus-S2 spectrum exhibits a marked reduction in the number/intensity of vibrational bands, compared to Ca PSII. A decrease in the amount of charge separation in Sr PSII cannot account for this observation. Comparison of Sr PSII ferricyanide/ferrocyanide band intensities (2116/2038 cm−1) before correction revealed ∼80% intensity with respect to Ca PSII upon each flash (data not shown). This comparison demonstrates that charge separation occurs in Sr PSII with high yield upon this second actinic flash. The spectra in Figure 3 are corrected for this small difference in ferricyanide/ferrocyanide band intensities. Note that while the 1900−1200 cm−1 regions are distinct in Ca PSII and Sr PSII, the W5+ water band region was not altered (Figure 5). Significantly, when Ca PSII (red) and Sr PSII (black) were compared, the S0-minus-S3 (third flash, Figure 3C) and S1minus-S0 spectra (fourth flash, Figure 3D) were similar. Confirming this conclusion, the Ca-minus-Sr double difference spectra (Figure 4C and D) for these transitions were similar to the negative control (Figure 4E). The high-frequency regions of the data were also similar (Figure 4C and D). Thus, the Srdependent decrease in intensity is specific to the 1600−1200 cm−1 region of the S2 to S3 transition. Vibrational bands in the 1800−1200 cm−1 region of a protein spectrum arise primarily from the peptide bond.42 Amino acid side-chain vibrational bands are generally less intense (see refs 46 and 47). However, amino acid side chains, such as carboxylate groups or carboxylate ligands, may also be observed under some conditions.39 The OEC is bound primarily by carboxylate groups.7,17 For example, bands at (negative) 1560/ 1403 and (positive) 1587/1364 cm−1 have previously been attributed to a bridging carboxylate ligand in the OEC.48 If observed, carboxylate ligand vibrations are expected to be sensitive to the coordination type and charge on the metal, but the frequency ranges for each ligand type and oxidation state overlap.40,49 For example, a protonated carboxylic acid exhibits 1720−1700 cm−1 (CO) and 1200 cm−1 (C−O) bands, while deprotonated carboxylates have delocalized asymmetric and symmetric stretching vibrations at ∼1560 and ∼1390 cm−1, respectively.50 When bound to a metal, carboxylate band frequencies can be unaffected, or the splitting between the asymmetric and symmetric stretching vibrations can increase. In a study of high-valent, multinuclear Mn compounds, coordination to higher-valence manganese caused a decrease in splitting between the asymmetric and stretching vibrations of bridging carboxylate ligands even without a change in coordination geometry.40 To be observed in the reactioninduced FT-IR spectrum, bands from the peptide backbone or,

mid-infrared spectrum is distinct from one reported previously in cyanobacterial PSII,43 most likely due to structural differences between plants and cyanobacteria.5,33 Figure 5

Figure 5. Infrared band from the protonated water cluster (W5+) as observed in reaction-induced FT-IR spectra at 263 K. Spectra were acquired from Ca PSII (red) or Sr PSII (black). (A) S2-minus-S1 spectrum, (B) S3-minus-S2 spectrum, (C) S0-minus-S3 spectrum, and (D) S1-minus-S0 spectrum in Ca PSII or Sr PSII. In (A−D), the gray spectrum was acquired from calcium-depleted PSII with a single flash. In (E), a baseline collected prior to the first flash (S1-minus-S1) is shown. Y-axis tick marks represent 1 × 10−4 absorbance units. Spectra are averages of data from 31 (A−D, red), 16 (A−D, black), 15 (A−D, gray), and 16 (E, black) samples.

presents the 3150−2200 cm−1 region of the S2-minus-S1 FT-IR spectrum. As reported previously, a band from the W5+ water cluster is observed in Ca PSII (red) but is not significant in Sr PSII (black), relative to a calcium-depleted control (gray). This difference between Ca PSII and Sr PSII was attributed previously to a change in the size of the water cluster in Sr PSII.35,44,45 In particular, broadening would make the band more difficult to detect in Sr PSII. 3359

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possibly, amino acid side chains must be altered by the oxidation of the Mn4CaO5 cluster. In these studies, we report that the S3-minus-S2 spectra are dramatically altered when Sr PSII is compared to Ca PSII. One possible explanation is a unique shift in coordination geometry when the Sr S2 and S3 states are compared. Carboxylate shifts are proposed to accompany catalysis in other metalloproteins (see ref 51 and references therein). Comparison of multiple structures of the OEC reveals plasticity in the positions of some carboxylate ligands, although these structures were determined at different resolutions (Figure S1, Supporting Information). A unique charge distribution in the Sr S3 state, when compared to the Ca S3 state, is a possible determining factor. For example, if a Mn oxidation reaction occurs upon this transition, the oxidation may be delocalized as a Mn(IV)O with high spin on the oxygen, as suggested by density functional theory (DFT) calculations.52,53 Such a delocalized charge could be responsive to the expected change in electronegativity when Sr is compared to Ca. Recent resonant inelastic X-ray scattering studies have indicated that charge is likely to be highly delocalized in the OEC, relative to the charge distribution in high-valent Mn model compounds.54 In recent PSII structures, there are six carboxylate (D170-D1, E189-D1, D342-D1, E333-D1, E354-CP43, and the carboxyterminus of A344-D1) and one histidine (H332-D1) ligand to the Mn4Ca(Sr)O5 cluster.7,17 Of these, two amino acid residues, D170-D1 and A344-D1, are predicted to bridge Ca and Mn.7,17 E189-D1 is predicted to be a unidentate ligand to Mn; however, E189 is only 3.4 Å away from Ca,7 and therefore, E189 could provide calcium ligation in some part of the S state cycle. Such an S-state-triggered alteration in the mode of ligation could explain our results if these conformational dynamics occur in Ca PSII but are not accessible in Sr PSII. Carboxylate ligation shifts were suggested from studies of the S1 to S2 transition in the D170E-D1 mutant.55 In particular, it was proposed that a bridging/bidentate carboxylate ligand shifted to unidentate ligation in this mutant. However, such shifts were not reported in the D170H-D1 mutant at higher temperature.56,57 See the Supporting Information for a more detailed description. In addition to a possible contribution from carboxylate side chains, these spectra will also reflect alterations in amide backbone hydrogen bonding. Our data suggest that there is conformational flexibility in the OEC. Structural plasticity, involving multiple, almost equienergetic states, has been suggested previously, on the basis of DFT calculations.52,53 For example, small changes in oxygen metal distance may account for the observation of the g = 4.1 and 2.0 S2 EPR signals (see the review in ref 5 and references therein). Our data show that the substitution of strontium for calcium has a dramatic structural impact on the S2 to S3 transition, although both Ca and Sr PSII oxidize water and produce molecular oxygen. Our data suggest that the wateroxidizing complex has access to at least two active conformational substates along its reaction coordinate.

Letter

ASSOCIATED CONTENT

S Supporting Information *

Description of experimental methods, including the sample purification strategy, strontium substitution, oxygen evolution activity measurements, and acquisition parameters for reactioninduced FTIR spectroscopy experiments, previous site-directed mutagenesis studies, and PSII structures (Figure S1). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Address: Department of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332-0400. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSF MCB 08-42246 (to B.A.B.). ABBREVIATIONS DFT density functional theory ENDOR electron nuclear double resonance EPR electron paramagnetic resonance EXAFS extended X-ray absorption fine structure FT-IR Fourier transform infrared PSII photosystem II OEC oxygen-evolving complex XANES X-ray absorption near-edge spectroscopy

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REFERENCES

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EXPERIMENTAL METHODS See the Supporting Information for experimental methods, including the sample purification strategy, strontium substitution, oxygen evolution activity measurements, and acquisition parameters for reaction-induced FTIR spectroscopy experiments. 3360

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The Journal of Physical Chemistry Letters

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