Calcium, conformational selection, and redox-activetyrosine YZ in the photosynthetic oxygen-evolving clusterZhanjun Guoa,b, Jiayuan Hea,b, and Bridgette A. Barrya,b,1

aSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332; and bPetit Institute of Bioengineering and Bioscience, GeorgiaInstitute of Technology, Atlanta, GA 30332

Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved April 17, 2018 (received for review January 17, 2018)

In Photosystem II (PSII), YZ (Tyr161D1) participates in radicaltransfer between the chlorophyll donor and the Mn4CaO5 cluster.Under flashing illumination, the metal cluster cycles among five Snstates, and oxygen is evolved from water. The essential YZ is tran-siently oxidized and reduced on each flash in a proton-coupledelectron transfer (PCET) reaction. Calcium is required for function.Of reconstituted divalent ions, only strontium restores oxygenevolution. YZ is predicted to hydrogen bond to calcium-boundwater and to His190D1 in PSII structures. Here, we report a vibra-tional spectroscopic study of YZ radical and singlet in the presenceof the metal cluster. The S2 state is trapped by illumination at190 K; flash illumination then generates the S2YZ radical. Usingreaction-induced FTIR spectroscopy and divalent ion depletion/substitution, we identify calcium-sensitive tyrosyl radical and ty-rosine singlet bands in the S2 state. In calcium-containing PSII, twoCO stretching bands are detected at 1,503 and 1,478 cm−1. Thesebands are assigned to two different radical conformers in calcium-containing PSII. At pH 6.0, the 1,503-cm−1 band shifts to 1,507 cm−1

in strontium-containing PSII, and the band is reduced in intensityin calcium-depleted PSII. These effects are consistent with ahydrogen-bonding interaction between the calcium site and oneconformer of radical YZ. Analysis of the amide I region indicatesthat calcium selects for a PCET reaction in a subset of the YZ con-formers, which are trapped in the S2 state. These results supportthe interpretation that YZ undergoes a redox-coupled conforma-tional change, which is calcium dependent.

water oxidation | photosynthesis | manganese–calcium cofactor |vibrational spectroscopy | radical

In plants, cyanobacteria, and algae, Photosystem II (PSII) car-ries out light-driven photosynthetic oxygen evolution (Fig. 1A).

This reaction is essential for the maintenance of aerobic life onearth and is a model in the development of sustainable, alter-native energy sources. Absorption of light by PSII induces acharge separation between the dimeric chlorophyll (chl) donor,P680, and a bound plastoquinone, QA or QB (Fig. 1A) (1, 2).P680

+ subsequently oxidizes Tyr161D1 (YZ), which then removesan electron from the nearby Mn4CaO5 cluster. The Mn4CaO5cluster is the site of water binding and oxidation. The YZ radical/YZ singlet redox couple acts as the interface between the one-electron chemistry of the reaction center and the four-electron–four proton reaction, which is required to release oxygen fromwater. On each light-driven step, YZ, which is tyrosine in theD1 polypeptide (Tyr161-D1, Fig. 1B) is transiently oxidized andreduced by a proton-coupled electron transfer (PCET) mechanism(Fig. 1C). YZ is essential for oxygen evolution. In X-ray struc-tures from cyanobacteria and cryo-EM structures from higherplants, YZ is hydrogen-bonded to His190 in the D1 poly-peptide. YZ is also hydrogen-bonded to a calcium-bound watermolecule (Fig. 1B) (1–5). The extensive, calcium-dependenthydrogen-bonding network has been proposed to be impor-tant in PCET reactions (6–9).

With flash illumination, the oxygen-evolving complex (OEC)cycles through five different oxidation states, named the Snstates, where n = 0–4 (10). The S1 state is the dark-stable state,and oxygen is produced during the S3-to-S0 transition (Fig. 1C).Cryogenic temperatures inhibit oxygen evolution by blockingsome of these transitions. At 190 K, the S1-to-S2 transition canoccur, but the other S-state transitions cannot. When a flash isgiven to the S2 state at this temperature, YZ is oxidized by P680

+,but, subsequently, YZ radical fails to oxidize the OEC (11).The QA

− to QB transition is also inhibited at 190 K (12).Therefore, in the 190 K trapped S2 state, a laser flash generatesthe YZ radical and QA

− state, which recombines via a PCETreaction (9, 13, 14).The role of calcium in the YZ PCET reactions has not been

fully elucidated. It is known that the decay time and midpointpotential of YZ radical are altered by manganese removal (forexamples, see refs. 15 and 16). From X-ray and cryo-EM struc-tures, calcium depletion is also expected to change hydrogen-bonding interactions between bound water molecules and YZ(1–5) (Fig. 1B). Calcium depletion can be readily reversed by thereaddition of calcium alone (reviewed in ref. 17). Althoughcalcium depletion is expected to change hydrogen bonding towater (18, 19), substantial changes in manganese ligation andmanganese–-manganese distances have not been detected incalcium-depleted samples (3, 20, 21). Among the divalent ions,only reconstituted strontium is able to replace calcium andsupports oxygen evolution activity (reviewed in refs. 22, 23).From an X-ray structure, strontium is known to bind at the

Significance

Photosynthetic water oxidation provides oxygen and maintainsaerobic life on earth. The Mn4CaO5 cluster is the catalytic site forthis reaction in Photosystem II. During water oxidation, a redox-active tyrosine, YZ, is oxidized and reduced in a proton-coupledelectron transfer reaction (PCET). We provide spectroscopic evi-dence that the essential calcium ion interacts with YZ in itsradical and singlet states. In addition, multiple conformationalstates of the YZ radical/singlet are identified. Calcium is shownto select for a PCET reaction in a subset of these states. Con-formational targeting could be a key to control of the PCETpathway and may occur in other enzymes, such as ribonucleo-tide reductase, which use tyrosine as a PCET cofactor.

Author contributions: B.A.B. designed research; Z.G. and J.H. performed research; Z.G.and B.A.B. analyzed data; and Z.G. and B.A.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: bridgette.barry@chemistry.gatech.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1800758115/-/DCSupplemental.

Published online May 11, 2018.

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calcium site (24). While there are no large changes compared withcalcium-containing PSII, this structure does exhibit a slightlyelongated Sr–water distance (24). Barium, with its larger ionicradius, is not effective in restoring oxygen evolution (9, 25), andhas been proposed to be a noncompetitive inhibitor of water ox-idation (26) and to disrupt hydrogen-bonding interactions in theproton transfer network (9).Here, we study the structure and interactions of YZ radical

and YZ singlet using reaction-induced Fourier transform in-frared (RIFT-IR) spectroscopy (27). The bands of radical andsinglet are distinguishable, due to the expected dramatic changesin force constants caused by oxidation of the aromatic ring (28).Notably, this is a report of the vibrational spectrum, YZ radicalminus YZ singlet, in an oxygen-evolving PSII preparation. Toslow the decay of YZ radical, cryogenic conditions were used totrap the OEC in the S2 state.

ResultsSamples. PSII preparations were purified and characterized bymethods described in SI Appendix, Fig. S1). PSII was depleted ofcalcium, and this preparation was used for CD-PSII experimentsat pH 6.0 and 7.5. This preparation was also used as the “parent”preparation for all reconstitution experiments. This preparationwas reconstituted either with strontium or calcium, generatingSr-PSII and Ca-PSII, respectively. The preparation was alsotreated with barium to give Ba-PSII.

Spectroscopic Methods. In the presence of an intact metal cluster,YZ radical is generated by a flash on the nanosecond time scale(via oxidation by P680

+) and reduced on the microsecond-to-millisecond time scale by the Mn4CaO5 cluster (29). To pro-long the lifetime of YZ radical and to obtain the YZ radical-minus-singlet spectrum in the presence of the Mn4CaO5 cluster,we first trapped the S2 state by continuous illumination at 190 K(Fig. 2A). This approach has been used in our published EPRstudies to generate the S2 state and to monitor the decay kineticsof YZ radical after a laser flash (SI Appendix, Fig. S2) (9, 13, 14).In the experiment described here, which monitors the RIFT-

IR signal of YZ radical, the experiment was performed similarlyto the published EPR studies. A 532-nm flash was given to the S2trapped sample (Fig. 2B) after a dark adaptation. This flashgenerates YZ·QA

− (Fig. 2C), as detected by the EPR controlexperiments (Fig. 2 C and D and SI Appendix, Fig. S2); thisspecies decays on the seconds time scale. For RIFT-IR studies,the decay of YZ·QA

− was monitored for a total of 90 s (Fig. 2D).To emphasize vibrational contributions from YZ radical, IR datawere recorded in 3-s time blocks. These blocks were used togenerate the difference RIFT-IR spectrum; for example, 3 s attime point B1 (0 s) and 3 s at time point B30 (87 s) (B1-minus-

B30, Fig. 2B). These data are expected to reflect a YZ radicalcontribution because the control EPR transients, recorded underthe same conditions, provide evidence for YZ radical decay onthis time scale (Fig. 2D and SI Appendix, Fig. S2) (9). The decayof the S2 state is minimal under these conditions. Further, QA

−

to QB transfer is blocked at this temperature (12). For moreinformation, see methods described previously (9, 13, 14, 30). Asan additional time point, data obtained in B10 (30 s) and B30(87 s) were also used to construct a difference RIFT-IR spec-trum from Ca-PSII. This second difference spectrum, B10-B30,is expected to reflect additional YZ radical decay after the flash,compared with B1-minus-B30.

RIFT-IR Spectra Associated with the Production of the S2 State, withYZ·QA

− Recombination in the S2 State, and Comparison with Controls.SI Appendix, Fig. S3A presents the S2QA

−-minus-S1QA spectrumobtained from Ca-PSII at pH 6.0, according to the method inFig. 2A. The S2QA

−-minus-S1QA spectra are characteristic of thistransition and have been reported previously under these con-ditions (30). Any YZ radical contribution to these S2QA

−-minus-S1QA spectra will be minimal because there is an ∼2-min darkadaptation time after the red illumination is discontinued. Thisdark time allows time for YZ radical to decay. The YD radicalcontribution is minimized due to a preflash, which is given atroom temperature before the sample is cooled to 190 K. Thepreflash is given to minimize the YD radical contribution andassist in synchronization in the S1 state.SI Appendix, Fig. S3B presents spectra obtained of YZ·QA

−-minus-YZQA (Fig. 2B, B1-minus-B30), as recorded from the S2state at 190 K. The spectrum in SI Appendix, Fig. S3B has beenexpanded by a factor of 5 relative to SI Appendix, Fig. S3A forcomparison purposes. As shown, in some regions of the mid-IRspectrum, the S2- and YZ-associated difference spectra areeasily distinguishable by comparing the relative intensities ofvibrational bands. For example, bands at 1,606, 1,324, and1,301 cm−1 are characteristic of YZ·QA

−-minus-YZQA and arenot observed in the S2QA

−-minus-S1QA spectrum. The relativeintensity differences at 1,718, 1,579, 1,554, and 1,534 cm−1 also

Fig. 1. (A) Redox cofactors in PSII. In the OEC, manganese atoms are shownin purple, calcium is shown in green, and oxygens are shown in red [ProteinData Bank (PDB) ID code 4UB6]. (B) YZ and its hydrogen-bonding partners.Water is represented by blue spheres (PDB ID code 4UB6). Illustration ofdivalent ion substitution and calcium depletion, starting with Ca-PSII (Sr2+ isdepicted in cyan sphere, and Ba2+ in yellow). (C) S-state cycle of OEC; theblack arrow indicates the only S-state transition allowed at 190 K. A proton-release pattern is shown (58). (Inset) YZ is oxidized and reduced by a PCETreaction.

Fig. 2. Monitoring the YZ·QA− recombination reaction in the S2 state. (A) The

S2 state is generated by continuous, red (633-nm) illumination at 190 K. RIFT-IRdata collection for the S1-to-S2 state transition is illustrated. Each data block is15 s, and the S2QA

−-minus-S1QA spectrum is generated as a control, P6-minus-D6. (B) A laser flash given to the S2 state at 190 K generating the YZ radical.RIFT-IR data collection monitoring the YZ·QA

− recombination reaction is il-lustrated. Each data block is 3 s, and the YZ·QA

−-minus-YZQA spectrum isgenerated, B1-minus-B30. To probe the reaction at later times, B10-B30 is used.There were 15 flashes and 15 rounds of rapid-scan FTIR data acquisition persample. (C) Representative EPR spectrum of S2YZ· in Ca-PSII. Black arrow in-dicates the field position used to monitor YZ·QA

− recombination in D. (Inset)Structure of tyrosine radical. (D) Representative EPR transient data monitoringYZ· in Ca-PSII at pH 6.0. The boxes show the data blocks and time scales usedto construct YZ·QA

− RIFT-IR spectra (B1-minus-B30; B10-minus-B30).

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distinguish the YZ·QA−-minus-YZQA from the S2QA

−-minus-S1QA spectrum.The spectrum in SI Appendix, Fig. S3B (Ca-PSII) is repeated in

Fig. 3F and compared with a spectrum acquired from CD-PSII(Fig. 3E) in the 1,600–1,400-cm−1 region. Importantly, YZ radicalyield and decay are similar in these two preparations, as assessed byEPR control experiments (SI Appendix, Fig. S2). Therefore, Fig. 3E and F are both expected to contain ring and CO stretching modesof YZ radical. In model tyrosyl radicals generated by 77K UVphotolysis and detected by FT-IR spectroscopy, unique radicalbands are observed at 1,577 (ring stretch), 1,516 (CO stretch), and1,448 (ring stretch) cm−1 (28). The CO stretch is at 1,514 cm−1, asdetected by Raman spectroscopy in solution (31, 32). These bandsare shifted in position from their frequencies in the singlet state andthus will appear in the difference spectra as unique positive signals.In CD-PSII at pH 6.0 (Fig. 3E), spectra resemble data

recorded using red illumination from manganese-depleted PSII,which cannot oxidize water, at higher temperature. See ref. 27for a discussion. In previous work, selective isotopic labeling ofQA

− or YZ radical showed that both species contribute in the1,480-cm−1 region (33, 34). To assess their contributions underthe conditions used here, SI Appendix, Fig. S4B presents a con-trol experiment using hydroxylamine to trap QA

−. In this ex-periment, hydroxylamine was added to Ca-PSII, and a laser flashwas given to a dark-adapted sample. This experiment is expectedto accumulate QA

−, and YZ radical formation is blocked underthis condition (35). In SI Appendix, Fig. S4B, a band at 1,484 cm−1

is observed. Previously, isotopic labeling of plastoquinone, density-functional theory (DFT) calculations, and RIFT-IR spectroscopyat 80 K led to the assignment of a 1,482-cm−1 band to the ring CCantisymmetric stretch of QA

− (33). Observation of a 1,484-cm−1

band in SI Appendix, Fig. S4B is, therefore, in agreement with thatprevious isotopic labeling work.A control spectrum, associated with YZ radical decay, was

also obtained from manganese-depleted (Tris-washed) PSII (SI

Appendix, Fig. S4 C–E) using 532-nm laser flashes. Comparisonof SI Appendix, Fig. S4 C–E follows the decay of the signal as afunction of time. As expected, the manganese-depleted spectrumis distinct in some regions from the intact S2YZ radical spectrum(compare SI Appendix, Fig. S4 A and C). However, both spectraexhibit a 1,478-cm−1 band. A band with this frequency was pre-viously assigned to the CO vibrational band of YZ radical byselective isotopic labeling of tyrosine and kinetic analysis (36–39). Taken together, these control experiments support the useof previous isotopic labeling assignments in the 1,480-cm−1 re-gion under these conditions.In Ca-PSII, the pattern of bands, detected in the RIFT-IR

spectrum, supports assignment to tyrosyl radical reduction aftercomparison with model compounds (SI Appendix, Fig. S3B).Bands are observed in regions of the spectrum corresponding tothe CO stretching vibrations of the radical (∼1,520–1,470 cm−1)and of the singlet (1,270–1,240 cm−1). This large frequency shiftis due to the delocalization of spin density in the radical (28).The appearance of amide I bands is also diagnostic of radicalformation in a peptide or protein (40). These regions of thespectrum are discussed below.

CO Stretching Bands of the YZ Radical at pH 6.0 and 7.5. As shown inFig. 3 A–D, removal of calcium and replacement with othercations does not have a significant effect on the spectrum associ-ated with S2QA

−-minus-S1QA (see additional spectral regions, SIAppendix, Fig. S5). However, in the YZ·QA

−-minus-YZQA spec-trum derived from Ca-PSII (Fig. 3F), a positive band is observedat 1,503 cm−1, which is calcium-sensitive. For example, the in-tensity of this band decreases when calcium is depleted (Fig. 3E).This is attributed to broadening, due to conformational flexibilityof the radical in the absence of calcium (see discussion of amide Iregion below). Further, strontium replacement (Fig. 3G) shifts thispositive band to 1,507 cm−1. In barium-treated PSII (Fig. 3H), thepositive band is observed at 1,512 cm−1. The frequency of thisband in barium-treated PSII is similar to the frequency in a modelcompound, i.e., tyrosyl radicals produced by UV photolysis of theamino acid in solution, 1,514 ± 2 cm−1 (32).Experiments were also conducted at pH 7.5 (SI Appendix, Fig.

S6). At this pH, CD-PSII and Ba-PSII exhibit bands with fre-quencies, which are similar, at 1,507 and 1,508 cm−1. This simi-larity between calcium-depleted and barium-treated preparationsis consistent with the proposed role of barium as a noncompeti-tive inhibitor (26) and with disruption of hydrogen-bonding in-teractions when barium interacts with PSII (9, 25). At pH 7.5,calcium reconstitution gives a broad band at 1,503 cm1, which isthe same frequency observed in Ca-PSII at pH 6.0. Strontiumreconstitution and pH 7.5 give a band at 1,504 cm−1, down-shiftedfrom the pH 6.0 Sr-PSII result. This pH dependence is attributedto protonation of amino acid residues, such as His190D1, nearYZ at pH 6.0 (1–5). Based on frequency and the EPR controlexperiments, we assign the Ca-PSII 1,503 cm−1 to a υ7a stretchingmode of YZ radical, in the presence of the Mn4CaO5 cluster andin one trapped conformational state. The observation of divalention-dependent shifts provides strong support for the assign-ment of the 1,503-cm−1 CO band to a calcium-interacting con-former of YZ.Based on previous isotopic labeling studies, the 1,478-cm−1

band (Fig. 3 E–H) is also assigned to the CO stretching mode ofthe YZ radical. This form of YZ radical is in a distinct envi-ronment due to an alteration in the trapped conformationalstate. The 1,478-cm−1 band is not calcium sensitive and appearsin the calcium-depleted PSII spectrum. This band is observedboth at pH 6.0 and 7.5 (SI Appendix, Fig. S6). This interpretationsuggests that there are at least two conformational states of YZradical, which differ in the degree of their interaction with thecalcium ion. Note that in the S2QA

−-minus-S1QA spectra (Fig. 3A–D), overlapping contributions from other redox active species,

Fig. 3. Spectra associated with the S1 to S2 transition (Left) and YZ·QA−

recombination (Right). RIFT-IR spectra (1,600–1,400 cm−1) were acquired at190 K and pH 6.0. In each panel, CD-PSII is black (A and E); Ca-PSII is red (Band F); Sr-PSII is blue (C and G), and Ba-PSII is green (D and H). (Left) Rep-resenting the S1-to-S2 transition, spectra are averages of 12 (A), 13 (B), 11 (C),and 14 (D). (Right) Representing YZ·QA

− recombination, a baseline, purple(I), is generated from S2-minus-S2, and spectra are averages of 17 (E), 15 (F),15 (G), 18 (H), and 12 (I) samples. Spectra were constructed as B1-minus-B30(Fig. 2D). The filled and marked bands are discussed in the text.

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amino acids, and peptide bonds contribute. While a completeassignment of the S2 spectrum at 190 K is not yet available (41),Fig. 3 A–D shows that calcium sensitivity is not observed inthis region.

CO Stretching Bands of YZ Singlet at pH 6.0. Fig. 4 (Left) presentsthe expanded region from 1,400 to 1,200 cm−1 of the YZ·QA

−-minus-YZQA spectrum. This region is predicted by modelcompound studies to contain a negative band, correspondingto the CO stretching mode of the YZ singlet state. TheCO stretching mode of tyrosine absorbs between 1,260 and1,240 cm−1, depending on the strength and type of hydrogenbonding to the phenolic oxygen (42). A negative band is observed at1,245 cm−1 in Ca-PSII (Fig. 4B), but is absent in CD-PSII (Fig. 4A).In Sr-PSII (Fig. 4C), the spectra are complex in this region, but inBa-PSII (Fig. 4D) two bands are observed. These data supportassignment of a calcium-dependent 1,245-cm−1 band to the YZsinglet state. The frequency is suggestive of a protonated YZ singletstate, which is hydrogen bonding as a proton acceptor (42). Whilebands are observed in this region in the S2QA

−-minus-S1QA spectra(SI Appendix, Fig. S5, Left), the S2/S1-derived bands are not iden-tical in frequency and not calcium dependent, and so can be dis-tinguished from the YZ singlet band. RIFT-IR spectra were alsoacquired at pH 7.5 and compared with the pH 6.0 spectra (SIAppendix, Fig. S7). The 1,245-cm−1 band is observed in Ca-PSIIeither at pH 6 or pH 7.5 (SI Appendix, Fig. S7 B and G).

Amide I Region. Fig. 4 (Right) shows the 1,750–1,600-cm−1 regionof the YZ·QA

− recombination spectrum; in this region, amide Ibands are expected at ∼1,650 cm−1. In model peptides and ri-bonucleotide reductase (RNR), oxidation/reduction of tyrosineis associated with amide I and II contributions to the spectrum(40, 43). These contributions are characteristic of sequence andwill also reflect the detailed backbone and dihedral angles as-sociated with the trapped conformer. The observation of twodifferent CO vibrational bands, as discussed above, is consistentwith the presence of at least two YZ radical conformers. All of

the spectra in Fig. 4 F–I exhibit negative, positive, negative bandsat 1,678, 1,667, and 1,660 cm−1, respectively, which can be at-tributed to a redox reaction in a conformer, with one defined setof backbone and dihedral angles (green fill). In Ba-PSII (Fig. 4I),the amide region exhibits these 1,678-, 1,667-, and 1,660-cm−1

bands and additional bands at positive 1,638 and negative1,630 cm−1 (dark-blue fill). These additional bands are consid-ered here as markers for a conformer, preferentially populatedin Ba-PSII. Comparison of spectra acquired in Ba-PSII with dataacquired in Sr-PSII (Fig. 4H) shows that while some of the amidebands are similar, Sr-PSII exhibits additional amide bands atpositive 1,653 and negative 1,648 cm−1 (light-blue fill). This re-flects a third conformational state, populated by strontiumbinding at the calcium site. In CD-PSII, the amide region of thespectrum contains amide bands corresponding to all of theconformers discussed above (Fig. 4F), attributable to increasedconformational flexibility, acquired in the absence of calcium.As described here, the amide I region of Ca-PSII is distinct

from that of Sr-PSII and Ba-PSII, when YZ·QA− data are

compared immediately following the laser flash (B1-minus-B30,Fig. 2). Notably, in Ca-PSII, additional amide I conformationalmarker bands appear at later times (B10-minus-B30 spectrum,Fig. 5B). These data show that calcium occupancy selects for aPCET reaction in a subset of YZ conformational states. Whenthe pH 6.0 experiments are compared with those recorded atpH 7.5 (SI Appendix, Fig. S8), the amide band frequencies areshifted, but the same overall patterns apply. For example, theamide I region of the CD-PSII spectrum appears to be the mostcomplex at pH 7.5, as also noted at pH 6.0.

Fig. 4. Divalent cation effects on spectra associated with YZ·QA− re-

combination. RIFT-IR spectra (1,400–1,200 cm−1, Left and 1,750–1,600 cm−1,Right) were acquired at 190 K and pH 6.0. Samples: CD-PSII is black (A and F);Ca-PSII is red (B and G); Sr-PSII is blue (C and H); Ba-PSII is green (D and I), andbaselines, purple (E and J), are generated from S2-minus-S2. Spectra are av-erages of 17 (A and F), 15 (B and G), 15 (C and H), 18 (D and I), and 12 (E andJ) samples. Spectra (A–D and F–I) were constructed as B1-minus-B30 (Fig. 2D).The filled bands are discussed in the text.

Fig. 5. Time dependence of the YZ·QA− RIFT-IR signal (Left) and a confor-

mational model (Right) showing divalent cation effects on the amide I re-gion. (Left) RIFT-IR spectra acquired at 190 K, showing YZ·QA

−-minus-YZQA

spectra from Ca-PSII at two different times after the laser flash. The spectraare (A) red, B1-minus-B30, corresponding to 0 s minus 87 s, and (B) brown,B10-minus-B30, corresponding to 30 s minus 87 s), at pH 6.0. See method,Fig. 2. Spectra in A and B are averages derived from 15 samples. C is abaseline, generated from S2-minus-S2 (12 samples). (Right) Calcium-dependent,conformational selection and the YZ·QA

− recombination reaction. Watersare shown as blue spheres (colors as Fig. 1). The conformer at the top is takenfrom PDB ID code 4UB6. The other conformers are speculative and weregenerated using the mutagenesis function.

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1,550- and 1,750-cm−1 Regions. In the YZ difference spectra, bandsare observed at 1,740–1,710 cm−1 (Fig. 4 and SI Appendix, Fig.S8). We attribute these bands to the transfer of Bohr protons toaspartate and glutamate residues in response to the YZ PCETreactions. In model compounds, the ν8a ring stretching vibra-tional band of the radical is expected as a positive band at1,556 cm−1 (28). A positive 1,554-cm−1 band in the YZ re-combination spectra (Fig. 3 E–H) has a frequency consistent witha radical ring stretching assignment. This band is observed at pH6.0 and 7.5 (SI Appendix, Fig. S6).

DiscussionBiological radical transfer reactions are important in many en-zymatic processes and are often facilitated by redox-active tyro-sines. In proteins such as PSII and RNR, conserved tyrosine sidechains act as electron transfer intermediates and reaction initi-ators, respectively. In PSII, YZ radical accepts an electron fromthe Mn4CaO5 cluster and drives transitions in the S-state cycle.The midpoint potential of tyrosine depends on pH (44). Thedriving force for each S-state transition is small (45). Therefore,the tyrosyl radical must be reprotonated on each electron transferto preserve the high-midpoint potential of YZ.To control the high reactivity of the radical state, a conforma-

tional control mechanism could be adopted. For example, atranslation of the aromatic ring, when the radical and singlet statesare compared, can make/break hydrogen bonds or change theaccessibility of the site to water. These changes can then be crucialto the mechanism, since the making and breaking of a hydrogenbond (∼3 kcal/mol) is significant in the energetics of PSII reac-tions. Redox-coupled structural changes have been proposed tooccur at two points in the RNR PCET pathway, Y122 and Y731(43, 46, 47). In addition, redox-coupled changes in secondarystructure have been observed and simulated in beta hairpin pep-tides (48). Thus, conformational gating could be a general methodof targeting and controlling PCET pathways in enzymes.This report provides a vibrational spectroscopic study of the YZ

radical and YZ singlet states in the presence of the Mn4CaO5cluster and a report of conformational selection in PSII. To obtainthis information, PSII sample was trapped in the S2 state, and therecombination of YZ radical and QA

− was monitored at 190 K.These are conditions in which YZ radical cannot oxidize the S2state, and the cycle is blocked. However, the metal cluster is intact,and relevant hydrogen-bonding interactions to the YZ radical andsinglet state are preserved. Using this approach, we present evi-dence for calcium-induced changes in the vibrational frequenciesof a subset of YZ radical and YZ singlet bands. In the presence ofcalcium, a positive band at 1,503 cm−1 is observed, assigned here toa conformer of YZ radical that interacts with the calcium ion. Theband at 1,503 cm−1 shifts to 1,507 cm−1 with strontium replacementand to 1,512 cm−1 after barium treatment. The 1,512-cm−1 fre-quency observed with barium treatment is similar to the frequencyobserved in model tyrosyl radical in solution with no divalent ioninteraction. This result is consistent with disruption of the hydro-gen-bond network after barium treatment, as previously proposed(9). The expected pKas of metal-bound water for calcium andstrontium are 12.7 and 13.2 (see refs. 22, 25, 26, and referencestherein). The observed shift when strontium-YZ (1,507 cm−1) iscompared with calcium-YZ (1,503 cm−1) is consistent with achange in hydrogen bonding between the YZ phenoxyl oxygen andcalcium/strontium-bound water. The intensity of the 1,503-cm−1

band is decreased in CD-PSII at pH 6.0, representing broadeningcaused by conformational flexibility at the YZ site induced in theabsence of calcium. At pH 7.5 in CD-PSII, the band is observed at1,507 cm−1, which is similar to the CO frequency observed in Ba-PSII at this pH value.For YZ singlet, divalent ion replacement or removal alters the

frequency of a negative band at 1,245 cm−1. In Sr-PSII at pH 6, thisband is shifted and reduced in intensity, attributable to the presence

of multiple interactions. In Ba-PSII, two bands are observed at1,263 and 1,234 cm−1. These results are consistent with a hydrogen-bonding interaction between the calcium site and the YZ singlet state.The YZ radical bands are down-shifted from those of tyrosyl

radical in solution, where the band is observed at 1,514 ± 2 cm−1

(28, 31, 32). However, in a matrix experiment, phenoxyl radical hasbeen reported to have a CO vibrational band at 1,481 cm−1 (49).This change was attributed to a decrease in the double-bondcharacter of the CO bond. Interactions between phenoxyl radicalsand metal complexes have been shown to down-shift the radicalCO frequency in model compounds (50). In Escherichia coli RNR,the Y122 radical has a CO stretching mode at 1,499 cm−1 (32, 51).The Y122 radical is not hydrogen bonded, but is located in a hy-drophobic environment (46). Therefore, the unique CO frequen-cies of YZ at 1,503 and 1,478 cm−1 (pH 6.0) radical reflecthydrogen bonding in a high dielectric environment and the uniqueinteractions of YZ with H190D1 and calcium-bound water. TheYZ singlet band at 1,245 cm−1 is characteristic of a strongly hy-drogen-bonded, protonated tyrosine, which accepts a hydrogenbond. Failure to observe the singlet CO band in CD-PSII and Sr-PSII is attributed to broadening and distribution of hydrogen-bonding interactions.One focus of our study is on the coupling of the YZ PCET

reactions with backbone and dihedral angle changes. This can beassessed from the amide I region. These data are consistent with acalcium-dependent selection mechanism. It has been shown pre-viously that oxidation of tyrosine in dipeptides (40), pentapeptides(52), and 18-mer beta hairpins (31) leads to appearance of amide Ivibrational bands in the RIFT-IR spectrum, associated with UVphotolysis and radical generation. For RNR, 13C1 isotopic labelingof the tyrosine backbone and DFT simulations led to the conclu-sion that the spectral pattern in the amide I and II regions is di-agnostic of the backbone and dihedral angle change. The B-radicalto A-singlet conformational change corresponded to an ∼100°change of backbone dihedral angle and was modeled to translatethe phenolic oxygen and make/break a hydrogen bond to D84 (43).To explain our PSII results, cryogenic illumination is proposed

to trap multiple conformers of the YZ-Mn4CaO5 cofactor. InCa-PSII, the pattern of bands in the amide I region is charac-terized by two negative bands at 1,678 and 1,660 cm−1. Thesemarker bands are observed in Sr-PSII, but Sr-PSII also exhibitsadditional amide bands. We hypothesize that these bands arecharacteristic of another YZ radical conformation. Distinctiveamide I marker bands appear in the Ca-PSII spectra on a longertime scale. Calcium depletion alters the amide region, suggestingthat interactions with calcium are important in conformer se-lection in the PCET reaction.In structures of PSII, YZ is hydrogen bonded to His190 in the

D1 polypeptide and to calcium-bound water (1–5). Our pre-vious EPR results led us to propose a two-pathway model forproton transfer to YZ radical (9). One, most likely dominant atlow pH, involves His190 in the D1 polypeptide. A secondpathway was proposed to involve water bound to calcium. Thesecurrent results are consistent with this picture and with hydro-gen bonding between calcium-bound water and YZ conformersboth in the radical and singlet states. Such an outcome is con-sistent with a role for this water in proton transfer. Our resultsalso provide evidence for a distinct local conformational changein the YZ side chain, which is controlled in Ca-PSII. Thisstrategy of a controlled, conformational rearrangement may beimportant in targeting PCET pathways in PSII and in otherredox enzymes, which conduct radical transfer with aromaticamino acid cofactors.

Experimental MethodsHighly active PSII was purified from market spinach using Triton X-100,followed by octylthioglucoside (OTG) treatment to give OTG-PSII samples(53, 54). The light-induced oxygen evolution rate (55) was greater than

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1,000 μmol O2 (mg chl·h)−1 at pH 6.0. Ethylene glycol-bis(β-aminoethylether)-N,N′,N′,N′-tetraacetic acid treatment at pH 7.5 was then used to gen-erate a CD-PSII sample (see SI Appendix, Fig. S1 for additional details). Cal-cium and strontium reconstitution (Ca-PSII and Sr-PSII) (9, 30), bariumtreatment (Ba-PSII) (9, 25, 26, 30), and Tris-treatment, generating manga-nese-depleted or “Tris-washed” PSII, were performed (56). EPR and RIFT-IR

spectroscopy were performed at pH 6.0 or pH 7.5 and 190 K (9, 13, 14, 30, 41,57). See SI Appendix.

ACKNOWLEDGMENTS. The authors thank Ms. Udita Brahmachari for helpfuldiscussions. The authors acknowledge National Science Foundation GrantMCB-14-11734 (to B.A.B.).

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