Fourier transform infrared difference spectroscopy for studying the molecular mechanism of photosynthetic water oxidation

The photosystem II reaction center mediates the light-induced transfer of electrons from water to plastoquinone, with concomitant production of O2. Water oxidation chemistry occurs in the oxygen-evolving complex (OEC), which consists of an inorganic Mn4CaO5 cluster and its surrounding protein matrix. Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been successfully used to study the molecular mechanism of photosynthetic water oxidation. This powerful technique has enabled the characterization of the dynamic structural changes in active water molecules, the Mn4CaO5 cluster, and its surrounding protein matrix during the catalytic cycle. This mini-review presents an overview of recent important progress in FTIR studies of the OEC and implications for revealing the molecular mechanism of photosynthetic water oxidation.


INTRODUCTION
Photosynthetic water oxidation is catalyzed by a Mn 4 Ca cluster and its surrounding protein matrix in photosystem II (PSII; Ferreira et al., 2004;Loll et al., 2005;Yano et al., 2006;Umena et al., 2011). The oxygen-evolving complex (OEC) accumulates oxidizing equivalents from the photochemical reactions within PSII and cycles through five oxidation states, termed S n (n = 0-4, n representing the storage of oxidizing equivalents in the OEC; Kok et al., 1970; Figure 1A). The molecule O 2 is produced in the transition of S 3 -(S 4 )-S 0 . One of the recent major breakthroughs in PSII research was the report of the crystal structure of oxygen-evolving PSII at 1.9 Å resolution . The structure of the Mn 4 CaO 5 cluster is shown in Figure 1B. Three Mn, one Ca, and four oxygen atoms form a cubane-like structure; the fourth Mn connects to the cubic structure by two μ-oxo-bridges. The Mn 4 CaO 5 cluster is connected with four water molecules: two are ligated to Ca and two to Mn 4  Figure 1B). These water molecules are candidates for substrates in photosynthetic water oxidation. Another distinct feature in the structure is the apparently longer bond distances between the O 5 -bridging oxygen atom and neighboring metal ions, which indicates weak bonding of this oxygen atom in the cluster. O 5 was proposed as a candidate for one of the substrates in dioxygen formation . More recent studies have suggested that the structure of the X-ray diffraction (XRD) model of PSII is modified by radiation-induced reduction of the Mn cluster (Luber et al., 2011;Grundmeier and Dau, 2012). Despite this problem, the 1.9 Å XRD structure is the crucial foundation for spectroscopic and mechanistic studies of photosynthetic water oxidation.
Fourier transform infrared difference spectroscopy has been widely used to study the structural changes in the OEC during the S-state catalytic cycle. The S 2 -minus-S 1 mid-frequency (1800-1000 cm −1 ) FTIR difference spectrum was first reported in 1992 (Noguchi et al., 1992). The S 3 -minus-S 2 spectrum of the PSII/OEC was reported in 2000 (Chu et al., 2000b), and spectra of flash-induced S-state transitions (S 1 → S 2 , S 2 → S 3 , S 3 → S 0 , and S 0 → S 1 ) during the complete S-state cycle were reported 1 year later (Hillier and Babcock, 2001;Noguchi and Sugiura, 2001). Many FTIR studies of the OEC focused on the midfrequency region (1800-1000 cm −1 ) of the IR spectrum, which contains information on structural changes of protein backbones and amino acid side-chains associated with S-state transitions of the OEC. One very important development in FTIR studies of the OEC were reports of high-frequency spectra (3700-3500 cm −1 ) of the OEC, which contain information on structural changes of the weakly H-bonded OH-stretching of active water molecules during S-state transitions of the OEC Sugiura, 2000, 2002a,b). The other important developments were reports of low-frequency spectra (<1000 cm −1 ), which contain information on metal-ligand and manganese-substrate vibration modes  . Images generated in the PyMOL program using Protein Data Bank entry 3ARC.
This mini-review gives an overview of recent important progress in FTIR studies of the OEC, combined with new spectroscopic and XRD structural information, to understand the chemical mechanism of photosynthetic water oxidation. More comprehensive reviews on FTIR studies of the OEC are available (Noguchi, 2007(Noguchi, , 2008aDebus, 2008).

OH-STRETCHING VIBRATIONAL MODES OF ACTIVE WATER MOLECULES IN THE HIGH-FREQUENCY REGION (3700-3500 cm -1 ) OF THE OEC
The reactions of substrate water during the S-state catalytic cycle of the OEC are of paramount importance to understand the chemical mechanism of photosynthetic water oxidation. In the new XRD structure of the Mn 4 CaO 5 cluster, the four water molecules connected to the OEC are involved in a hydrogen-bonded network linking the Mn 4 CaO 5 -cluster and Y Z . The bond distances (2.8-3.3 Å) between oxygen atoms of coordinated water molecules and their neighboring water molecules indicate that most of the O-H groups of the water molecules are weakly hydrogen bonded and will appear in the weakly hydrogen-bonded OH-stretch (3750-3500 cm −1 ) region of the FTIR spectra. Noguchi and colleagues reported flash-induced difference spectra of S-state transitions in the weakly H-bonded OH-stretching region Sugiura, 2000, 2002a,b). One active water molecule on the OEC, which gave rise to the S 1 band at ∼3585 cm −1 and the S 2 band at ∼3618 cm −1 , was identified at 250 K in light-induced S 2/ S 1 FTIR difference spectrum (Noguchi and Sugiura, 2000) and during the S-state cycle at 10 • C (Noguchi and Sugiura, 2002a,b) in PSII core complexes from Thermosynechococcus elongatus. The results indicated a weakened hydrogen bond of the OH group for one water molecule connected to the OEC during the S 1 → S 2 transition. In contrast to the S 1 → S 2 transition, the S 2 → S 3 , S 3 → S 0 , and S 0 → S 1 transitions all showed a negative OH-stretching mode at different frequencies, which indicates that these water (or hydroxide) molecules were involved in proton release reactions of the OEC or formed strong hydrogen-bonding interactions during these transitions (Noguchi and Sugiura, 2002a,b). In addition, these observations are consistent with a recent FTIR study which concluded that the proton release pattern from the substrate water on the OEC is in 1:0:1:2 stoichiometry for the S 0 → S 1 → S 2 → S 3 → S 0 transition ). One of the important issues is the exact location of these active water molecules detected by FTIR difference spectra on the OEC (e.g., associated with Mn or Ca) in the new XRD structure.

Mn-LIGAND AND Mn-SUBSTRATE VIBRATION MODES IN THE LOW-FREQUENCY REGION (<1000 cm -1 ) OF THE OEC
From studies of Mn model compound, Mn-ligand and Mnsubstrate vibration modes of the PSII/OEC are expected to show up in the low-frequency region (<1000 cm −1 ) of the IR spectrum (Chu et al., 2001c). In low-frequency S 2 /S 1 FTIR difference spectra of octyl-β-D-thioglucopyranoside (OTG) PSII core preparations of spinach, a positive mode at 606 cm −1 in 16 O water clearly downshifted to 596 cm −1 in 18 O water (Chu et al., 2000c; Figure 2A). With double-difference (S 2 /S 1 and 16 O minus 18 O) spectra, the 606 cm −1 mode was assigned to an S 2 mode, and a corresponding S 1 mode at about 625 cm −1 was identified (Chu et al., 2000c). In addition, this 606-cm −1 mode was up-shifted to about 618 cm −1 with Sr 2+ substitution but not significantly affected by 44 Ca isotope substitution (Chu et al., 2000c;Kimura et al., 2005a; Figure 2B). From these results and studies of Mn model compounds, this vibrational mode at 606 cm −1 in the S 2 state was assigned to a Mn-O-Mn cluster vibration in the OEC (Chu et al., 2000c). The structure of this Mn-O-Mn cluster very likely includes additional oxo and carboxylate bridges(s). IR modes for υ(Mn=O) and υ asy (Mn-O-Mn) for a singly oxobridged Mn cluster usually occur at >700 cm −1 and typically have a 30-40 cm −1 downshift (Chu et al., 2001c). They are unlikely to be the origin of the 606-cm −1 mode. Furthermore, this 606-cm −1 mode was altered in S 2 /S 1 FTIR difference spectra of Ala344D1Gly, Glu189Gln, and Asp170HisD1 Synechocystis mutant PSII particles (Chu et al., 2001a;Mizusawa et al., 2004;Kimura et al., 2005c). All the above amino acid residues are direct ligands for the Mn 4 Ca cluster. Therefore, the structure of the Mn-O-Mn cluster is structurally coupled to its surrounding ligand environment.
Low-frequency S 3 /S 2 spectra were reported in OTG PSII core preparations of spinach, in which intense bands at 604(−) and 621 (+) cm −1 were sensitive to 18 O water exchange (Chu et al., 2001b). The S 3 mode at ∼621 cm −1 was attributed to the Mn-O-Mn cluster mode of the S 3 state. Kimura et al. (2005b) reported on 16 O/ 18 O-and/or H/D water-sensitive low-frequency vibrations of the OEC during the complete S-state cycle in PSII core particles from T. elongatus. The S 2 mode at ∼606 cm −1 changed their sign and intensity during S-state cycling, which indicates Sstate-dependent changes in the core structure of the Mn 4 CaO 5 cluster. In addition, several IR bands sensitive to both 16 O/ 18 O and H/D exchanges were attributed to S-state intermediates during the S-state cycling (Kimura et al., 2005b). Furthermore, an intense 577(−) cm −1 band in the S 2 /S 1 spectra was found insensitive to universal 15 N-and 13 C-isotope labeling and assigned to the skeletal vibration of the Mn cluster or stretching vibrational modes of the Mn ligand (Kimura et al., 2003).
Low-frequency FTIR results demonstrate that one bridged oxygen atom in the Mn-O-Mn cluster of the OEC is accessible to and can be exchanged with bulky-phase water. This exchange occurs within minutes or faster because it is complete within 30 min (Chu et al., 2000c). A recent study involving W-band 17 O electronelectron double resonance-detected nuclear magnetic resonance (NMR) spectroscopy reported that one μ-oxo bridge of the OEC can exchange with H 2 17 O on a time scale (≤15 s) similar to that of substrate water on the OEC (Rapatskiy et al., 2012). This study also suggested that the exchangeable μ-oxo bridge links the outer Mn to the Mn 3 O 3 Ca open-cuboidal unit (O 4 and O 5 in Figure 1). The authors of this study favored the Ca-linked O 5 oxygen assignment (Rapatskiy et al., 2012). Low-frequency FTIR results showed that the Mn-O-Mn cluster mode at 606 cm −1 is sensitive to Sr 2+ substitution but not 44 Ca substitution (Chu et al., 2000c;Kimura et al., 2005a). Considering the structure of O 5 in the Mn 4 CaO 5 cluster  Figure 1B), the 44 Ca-induced isotopic shift of the Mn-O-Mn cluster mode may have been too small to be detected by previous FTIR studies. Thus, the O 5 -bridging oxygen atom is a good candidate for the exchangeable-bridged oxygen atom in the Mn-O-Mn cluster identified by FTIR. A recent continue-wave Q-band electron nuclear double resonance (ENDOR) study reported a much slower 17 O exchange rate (on the time scale of hours) with 17 O-labeled water into the μ-oxo bridge of the OEC (McConnell et al., 2011). Future study is required to resolve this discrepancy.

EFFECT OF AMMONIA ON THE OEC
Because of the structural similarity between NH 3 and H 2 O and the ability of NH 3 to inhibit photosynthetic water oxidation, the NH 3 binding site on the OEC might occur at the substrate waterbinding site. Previous EPR studies of NH 3 -treated PSII samples demonstrated that the S 2 -state multiline EPR signal is altered when samples illuminated at 200 K are subsequently "annealed" above 250 K (Beck et al., 1986;Britt et al., 1989). FTIR studies showed that NH 3 induced characteristic spectral changes in the S 2 /S 1 spectra at 250 K (Chu et al., 2004a;Fang et al., 2005). Among them, the S 2 -state symmetric carboxylate stretching mode at 1365 cm −1 in the S 2 /S 1 spectrum of control samples up-shifted to ∼1379 cm −1 in NH 3 -treated samples. This carboxylate mode was also altered by Sr 2+ substitution Suzuki et al., 2006), which indicates that the action site of NH 3 on the OEC is near the Ca 2+ site. In addition, the conditions that give rise to the NH 3 -induced up-shift of this S 2 -state carboxylate stretching mode at 1365 cm −1 are strongly correlated with those producing the modified S 2 -state multiline EPR signal (Chu et al., 2004a;Fang et al., 2005). Furthermore, a recent FTIR result showed that NH 3 did not replace the active water molecule connected to the OEC during the S 1 -to-S 2 transition at 250 K, whereas the Mn-O-Mn cluster vibrational mode at 606 cm −1 was diminished or underwent a large shift (Hou et al., 2011; Figure 2C). The above results are consistent with the proposal that NH 3 may replace one of the bridging oxygen atoms, presumably O 5 , in the Mn 4 CaO 5 cluster during the S 1 -to-S 2 transition (Britt et al., 1989). The other intriguing FTIR finding is that the effect of NH 3induced up-shift of 1365 cm −1 mode in the S 2 /S 1 spectrum was diminished at temperatures above 0 • C (Huang et al., 2008). The results indicate that the interaction of NH 3 with the OEC is attenuated at temperatures above 0 • C (Huang et al., 2008). In addition, a recent FTIR study reported an inhibitory effect of the ammonium cation on the PSII/OEC at 283 K (Tsuno et al., 2011). The results suggested that the ammonium cation perturbs some carboxylate residues coupled to the Mn cluster during the S 1 -to-S 2 transition and inhibits the oxygen evolution reaction at 283 K (Tsuno et al., 2011).

FTIR RESULTS FOR PROTEIN LIGANDS OF THE OEC
Fourier transform infrared studies involving isotopic labeling and site-directed mutagenesis have provided a wealth of information on dynamic structural changes of the protein backbones and amino acid side-chains during the S-state transitions of the OEC (Debus, 2008;Noguchi, 2008a;Shimada et al., 2011). An isotopeedited FTIR study identified the L-[1-13 C]alanine-sensitive symmetric carboxylate stretching modes in S 2 /S 1 difference spectra to the α-COO − group of D1-Ala344 (Chu et al., 2004b). This mode appears at ∼1356 cm −1 in the S 1 state and at ∼1339 or ∼1320 cm −1 in the S 2 state in unlabeled wild-type PSII particles but not in D1-Ala344Gly and D1-Ala344Ser mutant PSII particles. These frequencies are consistent with unidentate ligation of the α-COO − group of D1-Ala344 to the Mn 4 Ca cluster in both the S 1 and S 2 state (Chu et al., 2004b;Strickler et al., 2005). In addition, substituting Sr for Ca did not alter the symmetric carboxylate stretching modes of D1-Ala344 . The results suggested that the α-COO − group of D1-Ala344 did not ligate Ca. In the 1.9 Å XRD structure, the α-COO − group of D1-Ala344 shows very asymmetrical bridging between Mn 2 and Ca in the cluster, with the Mn-O distance 2.0 Å and Ca-O distance 2.6 Å . In addition, the isotopic bands for the α-COO − group of D1-Ala344 showed characteristic changes during S-state cycling (Kimura et al., 2005d). These results indicated that the C-terminal Ala 344D1 is structurally coupled, presumably directly ligated, to the Mn ion that undergoes oxidation of Mn(III) to Mn(IV) during the S 1 -to-S 2 transition and is reduced in reverse with the S 3 -to-S 0 transition (Chu et al., 2004b;Kimura et al., 2005d). In contrast, mutations of D1-Asp170, D1-Glu189, and D1-Asp342 did not eliminate any carboxylate vibrational stretching modes during S-state cycling of the OEC Strickler et al., 2006Strickler et al., , 2007. Recent computational studies suggested that vibrations of carboxylate ligands can be quite insensitive to Mn oxidation, if they are not coordinated along the Jahn-Teller axis (Sproviero et al., 2008). In their model, the only amino acid residue that is ligated along the Jahn-Teller axis of a Mn III ion is CP43-E354.
Of note, CP43-E354Q mutant PSII particles gave rise to characteristic spectral changes in the amide and carboxylate stretch regions of FTIR difference spectra during S-state transitions (Strickler et al., 2008;Shimada et al., 2009;Service et al., 2010). In addition, the weakly H-bonded O-H stretching modes of the active water molecule associated with the OEC were significantly altered in S 2 /S 1 FTIR difference spectra of CP43-E354Q mutant PSII particles (Shimada et al., 2009). Furthermore, H 2 18 O exchange mass spectrometry experiments showed that the CP43-E354Q mutation weakened the binding of both substrate-water molecules (or water-derived ligands), particularly affecting the one with faster exchange in the S 3 state (Service et al., 2010). The XRD structure of the OEC showed that coordinated water molecules were on Ca 2+ and Mn 4 , which were both not ligated by CP43-E354 . Presumably, CP43-E354Q mutation may induce significant structural changes to the Mn 4 CaO 5 core that affects associated active water molecule(s) on the OEC during the S 1 -to-S 2 transition.
A recent time-resolved infrared study revealed the proton and protein dynamics associated with the OEC during the S-state transitions (Noguchi et al., 2012). The results suggest that during the S 3 -to-S 0 transition, protons are greatly rearranged to form a transient state before the oxidation of the Mn 4 CaO 5 cluster that leads to O 2 formation. In addition, an early proton movement was detected during the S 2 → S 3 transition, indicating a proton release coupled with the electron transfer reaction. Furthermore, a relatively slow carboxylate movement occurred in the S 0 → S 1 transition, which might reflect the protein relaxation process to stabilize the S 1 state (Noguchi et al., 2012). This study demonstrates that time-resolved infrared technique is extremely useful to monitor proton and protein dynamics of the OEC during photosynthetic oxygen evolution.

BIOINORGANIC MODELS FOR FTIR SPECTRAL INTERPRETATION
Vibrational data from model compounds relevant to the OEC is crucial to interpret FTIR data of the OEC during S-state cycling. However, vibrational data for synthetic multinuclear Mn complexes are still limited (Cua et al., 2003;Berggren et al., 2012). Particularly, vibrational data are needed for the Ca-Mn multinuclear cluster that models the Mn 4 CaO 5 cluster (Kanady et al., 2011;Mukherjee et al., 2012). One previous study reported IR spectra and normal mode analysis of the adamantine-like complex [Mn 4 O 6 (bpea) 4 ] n+ (Visser et al., 2002). By using the electrochemical method to record the difference IR spectrum and 18

CONCLUSIONS AND PERSPECTIVES
Light-induced FTIR difference spectroscopy has become a fruitful structural technique to study the molecular mechanism of photosynthetic water oxidation. The new high-resolution XRD structure of the OEC has served as a crucial foundation for designing FTIR experiments and interpreting FTIR data. Combined with isotopic labeling, site-directed mutagenesis, model compound studies, and normal mode analysis, FTIR difference spectroscopy will continue to provide important structural and mechanistic insights into the water-splitting process in PSII.

ACKNOWLEDGMENTS
The author is grateful to Prof. Richard J. Debus for helpful suggestions on the manuscript. This work was supported by National Science Council in Taiwan (NSC 101-2627-M-001-001) and by Academia Sinica.