The mechanism for dioxygen formation in PSII studied by quantum chemical methods.

The availability of an X-ray structure for PSII including the water-oxidizing cluster, where the metal atoms and the amino acids are assigned, has opened up new possibilities to study the mechanism for dioxygen formation. In the present paper the main results of an ongoing hybrid DFT study are presented. The model used follows the structure suggested by the X-ray analysis as closely as possible. After nearly one thousand optimizations of different structures, each one with about 70 atoms, the main features of a water oxidizing mechanism start to emerge. The key intermediate is an oxyl radical state in S(3), stabilized by a weak trans effect to a bridging oxo in the cube. To reach this radical state a structural rearrangement appears necessary, in which one additional bridging oxo is formed between the dangling manganese and a manganese in the cube. The calculated energetics is reasonable but still not fully consistent with a correct mechanism. It is suggested that some part of the structure is not correct, probably the presence of the bicarbonate.     

Quantum chemical studies of manganese centers in biology

During the past five years, hybrid density functional theory has been used to study mechanisms for redox-active enzymes containing complexes with a variety of different transition metals. In this paper, suggested mechanisms of some manganese enzymes are described. For photosystem II, a mechanism is proposed leading to an oxyl radical in the S(3)-state, which is the precursor for the O-O bond formation. For manganese catalase, the suggested mechanism instead leads to the formation of a hydroxyl radical after the O-O bond of hydrogen peroxide is cleaved. This radical is immediately quenched by a manganese center. Parallels between these enzymes are highlighted. Jahn-Teller and trans effects are emphasized.     

A ligand field chemistry of oxygen generation by the oxygen-evolving complex and synthetic active sites

Oxygen-oxygen bond formation and O2 generation occur from the S4 state of the oxygen-evolving complex (OEC). Several mechanistic possibilities have been proposed for water oxidation, depending on the formal oxidation state of the Mn atoms. All fall under two general classifications: the AB mechanism in which nucleophilic oxygen (base, B) attacks electrophilic oxygen (acid, A) of the Mn4Ca cluster or the RC mechanism in which radical-like oxygen species couple within OEC. The critical intermediate in either mechanism involves a metal oxo, though the nature of this oxo for AB and RC mechanisms is disparate. In the case of the AB mechanism, assembly of an even-electron count, high-valent metal-oxo proximate to a hydroxide is needed whereas, in an RC mechanism, two odd-electron count, high-valent metal oxos are required. Thus the two mechanisms give rise to very different design criteria for functional models of the OEC active site. This discussion presents the electron counts and ligand geometries that support metal oxos for AB and RC O-O bond-forming reactions. The construction of architectures that bring two oxygen functionalities together under the purview of the AB and RC scenarios are described.     

QM/MM computational studies of substrate water binding to the oxygen-evolving centre of photosystem II

This paper reports computational studies of substrate water binding to the oxygen-evolving centre (OEC) of photosystem II (PSII), completely ligated by amino acid residues, water, hydroxide and chloride. The calculations are based on quantum mechanics/molecular mechanics hybrid models of the OEC of PSII, recently developed in conjunction with the X-ray crystal structure of PSII from the cyanobacterium Thermosynechococcus elongatus. The model OEC involves a cuboidal Mn3CaO4Mn metal cluster with three closely associated manganese ions linked to a single mu4-oxo-ligated Mn ion, often called the 'dangling manganese'. Two water molecules bound to calcium and the dangling manganese are postulated to be substrate molecules, responsible for dioxygen formation. It is found that the energy barriers for the Mn(4)-bound water agree nicely with those of model complexes. However, the barriers for Ca-bound waters are substantially larger. Water binding is not simply correlated to the formal oxidation states of the metal centres but rather to their corresponding electrostatic potential atomic charges as modulated by charge-transfer interactions. The calculations of structural rearrangements during water exchange provide support for the experimental finding that the exchange rates with bulk 18 O-labelled water should be smaller for water molecules coordinated to calcium than for water molecules attached to the dangling manganese. The models also predict that the S1-->S2 transition should produce opposite effects on the two water-exchange rates.     

Why did Nature choose manganese to make oxygen?

This paper discusses the suitability of manganese for its function in catalysing the formation of molecular oxygen from water. Manganese is an abundant element. In terms of its inherent properties, Mn has a particularly rich redox chemistry compared with other d-block elements, with several oxidizing states accessible. The most stable-state Mn2+ behaves like a Group 2 element--it is mobile, weakly complexing, easily taken up by cells and redox-inactive in simple aqueous media. Only in the presence of suitable ligands does Mn2+ become oxidized, so it provides an uncomplicated building unit for the oxygen-evolving centre (OEC). The intermediate oxidation states Mn(III) and Mn(IV) are strongly complexed by O2(-) and form robust mixed-valence poly-oxo clusters in which the Mn(IV)/Mn(III) ratio can be elevated, one electron at a time, accumulating oxidizing potential and capacity. The OEC is a Mn4CaOx cluster that undergoes sequential oxidations by P680+ at potentials above 1V, ultimately to a super-oxidized level that includes one Mn(V) or a Mn(IV)-oxyl radical. The latter is powerfully oxidizing and provides the crucial 'power stroke' necessary to generate an O-O bond. This leaves a centre still rich in Mn(IV), ensuring a rapid follow-through to O2.     

Probing the functional role of Ca2+ in the oxygen-evolving complex of photosystem II by metal ion inhibition

Photosynthetic oxygen evolution in photosystem II (PSII) takes place in the oxygen-evolving complex (OEC) that is comprised of a tetranuclear manganese cluster (Mn4), a redox-active tyrosine residue (YZ), and Ca2+ and Cl- cofactors. The OEC is successively oxidized by the absorption of 4 quanta of light that results in the oxidation of water and the release of O2. Ca2+ is an essential cofactor in the water-oxidation reaction, as its depletion causes the loss of the oxygen-evolution activity in PSII. In recent X-ray crystal structures, Ca2+ has been revealed to be associated with the Mn4 cluster of PSII. Although several mechanisms have been proposed for the water-oxidation reaction of PSII, the role of Ca2+ in oxygen evolution remains unclear. In this study, we probe the role of Ca2+ in oxygen evolution by monitoring the S1 to S2 state transition in PSII membranes and PSII core complexes upon inhibition of oxygen evolution by Dy3+, Cu2+, and Cd2+ ions. By using a cation-exchange procedure in which Ca2+ is not removed prior to addition of the studied cations, we achieve a high degree of reversible inhibition of PSII membranes and PSII core complexes by Dy3+, Cu2+, and Cd2+ ions. EPR spectroscopy is used to quantitate the number of bound Dy3+ and Cu2+ ions per PSII center and to determine the proximity of Dy3+ to other paramagnetic centers in PSII. We observe, for the first time, the S2 state multiline electron paramagnetic resonance (EPR) signal in Dy3+- and Cd2+-inhibited PSII and conclude that the Ca2+ cofactor is not specifically required for the S1 to S2 state transition of PSII. This observation provides direct support for the proposal that Ca2+ plays a structural role in the early S-state transitions, which can be fulfilled by other cations of similar ionic radius, and that the functional role of Ca2+ to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by Ca2+ and Sr2+, which have similar Lewis acidities.     

A comparison of the reaction mechanisms of iron- and manganese-containing 2,3-HPCD: an important spin transition for manganese

Homoprotocatechuate (HPCA) dioxygenases are enzymes that take part in the catabolism of aromatic compounds in the environment. They use molecular oxygen to perform the ring cleavage of ortho-dihydroxylated aromatic compounds. A theoretical investigation of the catalytic cycle for HPCA 2,3-dioxygenase isolated from Brevibacterium fuscum (Bf 2,3-HPCD) was performed using hybrid DFT with the B3LYP functional, and a reaction mechanism is suggested. Models of different sizes were built from the crystal structure of the enzyme and were used in the search for intermediates and transition states. It was found that the enzyme follows a reaction pathway similar to that for other non-heme iron dioxygenases, and for the manganese-dependent analog MndD, although with different energetics. The computational results suggest that the rate-limiting step for the whole reaction of Bf 2,3-HPCD is the protonation of the activated oxygen, with an energy barrier of 17.4 kcal/mol, in good agreement with the experimental value of 16 kcal/mol obtained from the overall rate of the reaction. Surprisingly, a very low barrier was found for the O-O bond cleavage step, 11.3 kcal/mol, as compared to 21.8 kcal/mol for MndD (sextet spin state). This result motivated additional studies of the manganese-dependent enzyme. Different spin coupling between the unpaired electrons on the metal and on the evolving substrate radical was observed for the two enzymes, and therefore the quartet spin state potential energy surface of the MndD reaction was studied. The calculations show a crossing between the sextet and the quartet surfaces, and it was concluded that a spin transition occurs and determines a barrier of 14.4 kcal/mol for the O-O bond cleavage, which is found to be the rate-limiting step in MndD. Thus the two 83% identical enzymes, using different metal ions as co-factors, were found to have similar activation energies (in agreement with experiment), but different rate-limiting steps.     

Recent pulsed EPR studies of the photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms

The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.     

Damage to the oxygen-evolving complex by superoxide anion, hydrogen peroxide, and hydroxyl radical in photoinhibition of photosystem II

Under strong illumination of a photosystem II (PSII) membrane, endogenous superoxide anion, hydrogen peroxide, and hydroxyl radical were successively produced. These compounds then cooperatively resulted in a release of manganese from the oxygen-evolving complex (OEC) and an inhibition of oxygen evolution activity. The OEC inactivation was initiated by an acceptor-side generated superoxide anion, and hydrogen peroxide was most probably responsible for the transportation of reactive oxygen species (ROS) across the PSII membrane from the acceptor-side to the donor-side. Besides ROS being generated in the acceptor-side induced manganese loss; there may also be a ROS-independent manganese loss in the OEC of PSII. Both superoxide anion and hydroxyl radical located inside the PSII membrane were directly identified by a spin trapping-electron spin resonance (ESR) method in combination with a lipophilic spin trap, 5-(diethoxyphosphoryl)-5-phenethyl-1-pyrroline N-oxide (DEPPEPO). The endogenous hydrogen peroxide production was examined by oxidation of thiobenzamide.     

Changes in structural and functional properties of oxygen-evolving complex induced by replacement of D1-glutamate 189 with glutamine in photosystem II: ligation of glutamate 189 carboxylate to the manganese cluster

A carboxylate group of D1-Glu-189 in photosystem II has been proposed to serve as a direct ligand for the manganese cluster. Here we constructed a mutant that eliminates the carboxylate by replacing D1-Glu-189 with Gln in the cyanobacterium Synechocystis sp. PCC 6803, and we examined the resulting effects on the structural and functional properties of the oxygen-evolving complex (OEC) in photosystem II. The E189Q mutant grew photoautotrophically, and isolated photosystem II core particles evolved oxygen at approximately 70% of the rate of control wild-type particles. The E189Q OEC showed typical S(2) state electron spin resonance signals, and the spin center distance between the S(2) state manganese cluster and the Y(D) (D2-Tyr-160), detected by electron-electron double resonance spectroscopy, was not affected by this mutation. However, the redox potential of the E189Q OEC was considerably lower than that of the control OEC, as revealed by the elevated peak temperature of the S(2) state thermoluminescence bands. The mutation resulted in specific changes to bands ascribed to the putative carboxylate ligands for the manganese cluster and to a few carbonyl bands in mid-frequency (1800 to 1100 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum. Notably, the low frequency (650 to 350 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum was also uniquely changed by this mutation in the frequencies for the manganese cluster core vibrations. These results suggested that the carboxylate group of D1-Glu-189 ligates the manganese ion, which is influenced by the redox change of the oxidizable manganese ion upon the S(1) to S(2) transition.     

Identification of a Mn-O-Mn cluster vibrational mode of the oxygen-evolving complex in photosystem II by low-frequency FTIR spectroscopy

We have developed conditions for recording the low-frequency S(2)/S(1) Fourier transform infrared difference spectrum of hydrated PSII samples. By exchanging PSII samples with buffered (18)O water, we found that a positive band at 606 cm(-)(1) in the S(2)/S(1) spectrum in (16)O water is clearly downshifted to 596 cm(-)(1) in (18)O water. By taking double-difference (S(2)/S(1) and (16)O minus (18)O) spectra, we assign the 606 cm(-)(1) mode to an S(2) mode and also identify a corresponding S(1) mode at about 625 cm(-)(1). In addition, by Sr and (44)Ca substitution experiments, we found that the 606 cm(-)(1) mode is upshifted to about 618 cm(-)(1) by Sr(2+) substitution but that this mode is not affected by substitution with the (44)Ca isotope. On the basis of these results and also on the basis of studies of Mn model compounds, we assign the 625 cm(-)(1) mode in the S(1) state and the 606 cm(-)(1) mode in the S(2) state to a Mn-O-Mn cluster vibration of the oxygen-evolving complex (OEC) in PSII. This structure may include additional bridge(s), which could be another oxo, carboxylato(s), or atoms derived from an amino acid side chain. Our results indicate that the bridged oxygen atom shown in this Mn-O-Mn cluster is exchangeable and accessible by water. The downshift in the Mn-O-Mn cluster vibration as manganese is oxidized during the S(1) --> S(2) transition is counterintuitive; we discuss possible origins of this behavior. Our results also indicate that Sr(2+) substitution in PSII causes a small structural perturbation that affects the bond strength of the Mn-O-Mn cluster in the PSII OEC. This suggests that Sr(2+), and by inference, Ca(2+), communicates with, but is not integral to, the manganese core.     

The S0 state of photosystem II induced by hydroxylamine: differences between the structure of the manganese complex in the S0 and S1 states determined by X-ray absorption spectroscopy

Hydroxylamine at low concentrations causes a two-flash delay in the first maximum flash yield of oxygen evolved from spinach photosystem II (PSII) subchloroplast membranes that have been excited by a series of saturating flashes of light. Untreated PSII membrane preparations exhibit a multiline EPR signal assigned to a manganese cluster and associated with the S2 state when illuminated at 195 K, or at 273 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). We used the extent of suppression of the multiline EPR signal observed in samples illuminated at 195 K to determine the fraction of PSII reaction centers set back to a hydroxylamine-induced S0-like state, which we designate S0*. The manganese K-edge X-ray absorption edges for dark-adapted PSII preparations with or without hydroxylamine are virtually identical. This indicates that, despite its high binding affinity to the oxygen-evolving complex (OEC) in the dark, hydroxylamine does not reduce chemically the manganese cluster within the OEC in the dark. After a single turnover of PSII, a shift to lower energy is observed in the inflection of the Mn K-edge of the manganese cluster. We conclude that, in the presence of hydroxylamine, illumination causes a reduction of the OEC, resulting in a state resembling S0. This lower Mn K-edge energy of S0*, relative to the edge of S1, implies the storage and stabilization of an oxidative equivalent within the manganese cluster during the S0----S1 state transition. An analysis of the extended X-ray absorption fine structure (EXAFS) of the S0* state indicates that a significant structural rearrangement occurs between the S0* and S1 states. The X-ray absorption edge position and the structure of the manganese cluster in the S0* state are indicative of a heterogeneous mixture of formal valences of manganese including one Mn(II) which is not present in the S1 state.     

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).     

The tetra-manganese complex of photosystem II during its redox cycle - X-ray absorption results and mechanistic implications

Using X-ray absorption spectroscopy (XAS), relevant information on structure and oxidation state of the water-oxidizing Mn complex of photosystem II has been obtained for all four semi-stable intermediate states of its catalytic cycle. We summarize our recent XAS results and discuss their mechanistic implications. The following aspects are covered: (a) information content of X-ray spectra (pre-edge feature, edge position, extended X-ray absorption fine-structure (EXAFS), dichroism in the EXAFS of partially oriented samples); (b) S(1)-state structure; (c) X-ray edge results on oxidation state changes; (d) EXAFS results on structural changes during the S-state cycle; (e) a structural model for the Mn complex in its S(3)-state; (f) XAS-based working model for the S(2)-S(3) transition; (g) XAS-based working model for the S(0)-S(1) transition; (h) potential role of hydrogen atom abstraction by the Mn complex. Finally, we present a specific hypothesis on the mechanism of dioxygen formation during the S(3)-(S(4))-S(0) transition. According to this hypothesis, water oxidation is facilitated by manganese reduction that is coupled to proton transfer from a substrate water to bridging oxides.     

Hydroxide instead of bicarbonate in the structure of the oxygen evolving complex

The energy diagram for the catalytic cycle of dioxygen evolution in photosystem II has been recomputed using a slightly different model than the one used previously, since the results showed an erroneous trend in the critical region from S(2) to S(3). By replacing the bicarbonate ligand, which was bridging between calcium and the outer manganese, by a hydroxide a significant improvement of the energy diagram is obtained. Most notably, the S(2) to S(3) transition is now exergonic as it should be. However, for the S(2)(0) state an artificial constraint is needed on the hydrogen bonding, indicating that there is still some problem with the model used. This hydroxide model was used to study the effects of replacing calcium with strontium, magnesium and cadmium. The computed results reproduce the results observed experimentally that magnesium and cadmium suppress water oxidation and that strontium slows it down. For both magnesium and cadmium the process stops already at S(2).     

pH dependence of the four individual transitions in the catalytic S-cycle during photosynthetic oxygen evolution

We have investigated the pH dependence for each individual redox transition in the S-cycle of the oxygen evolving complex (OEC) of photosystem II by electron paramagnetic resonance (EPR) spectroscopy. In the experiments, OEC is advanced to the appropriate S-state at normal pH. Then, the pH is rapidly changed, and a new flash is given. The ability to advance to the next S-state in the cycle at different pHs is determined by measurements of the decrease or increase of characteristic EPR signals from the OEC in different S-states. In some cases the measured EPR signals are very small (this holds especially for the S0 ML signal at pH >7.5 and pH <4.8). Therefore, we refrain from providing error limits for the determined pK's. Our results indicate that the S1 --> S2 transition is independent of pH between 4.1 and 8.4. All other S-transitions are blocked at low pH. In the acidic region, the pK's for the inhibition of the S2 --> S3, the S3 --> [S4] --> S0, and the S0 --> S1 transitions are about 4.0, 4.5, and 4.7, respectively. The similarity of these pK values indicates that the inhibition of the steady-state oxygen evolution in the acidic range, which occurs with pK approximately 4.8, is a consequence of similar pH blocks in three of the redox steps involved in the oxygen evolution. In the alkaline region, we report a clear pH block in the S3 --> [S4] --> S0 transition with a pK of about 8.0. Our study also indicates the existence of a pH block at very high pH (pK approximately 9.4) in the S2 --> S3 transition. The S0 --> S1 transition is not affected, at least up to pH 9.0. This suggests that the inhibition of the steady-state oxygen evolution, which occurs with a pK of 8.0, is dominated by the inhibition of the S3 --> [S4] --> S0 transition. Our results are obtained in the presence of 5% methanol (v/v). However, it is unlikely that the determined pK's are affected by the presence of methanol since our results also show that the pH dependence of the steady-state oxygen evolution is not affected by methanol. The results in the alkaline region are in good agreement with a model, which suggests that the redox potential of Y(Z*)/Y(Z) is directly affected by high pH. At high pH the Y(Z*)/Y(Z) potential becomes lower than that of S2/S1 and S3/S2. The acidic block, with a pK of 4-5 in three S-transitions, implies that the inhibition mechanism is similar, and we suggest that it reflects protonation of a carboxylic side chain in the proton relay that expels protons from the OEC.     

O-O bond cleavage and alkane hydroxylation in methane monooxygenase

Several new aspects of the O-O bond cleavage and alkane hydroxylation mechanisms have been studied by hybrid density functional theory in this reinvestigation of methane monooxygenase. As concerning key intermediates in these reactions, a new important low-lying state is found, described either as Fe2(III,V) or as Fe2(III,IV)O. A fully optimized transition state for O-O bond cleavage has been determined. It is suggested that the large difference in optimal size (as determined in gas phase) of the complex, before and after the O-O bond cleavage, leads to an additional driving force for the reaction, not considered previously. The strain of the enzyme is estimated to lead to a driving force in the forward direction of about 5 kcal/mol, which could explain some of the pH dependence found in recent experiments. For the hydroxylation reaction, a clean hydrogen abstraction transition state leading to a substrate radical is again found, in contrast to interpretations of radical clock experiments. An explanation, based on new results, is suggested that could account for both the experimental and theoretical results.     

Important roles of tyrosines in photosystem II and cytochrome oxidase

Theoretical studies (B3LYP) on models of the active sites in Photosystem II (PSII) and cytochrome oxidase are discussed. The role of a tyrosyl radical in the O-O bond formation in PSII is investigated, as well as the tyrosyl radical formation. In cytochrome oxidase, mechanisms for O-O bond cleavage involving tyrosyl radical formation are investigated, together with possible roles for the tyrosine in the proton translocation.     

Water oxidation by photosystem II: H(2)O-D(2)O exchange and the influence of pH support formation of an intermediate by removal of a proton before dioxygen creation

Understanding the chemistry of photosynthetic water oxidation requires deeper insight into the interrelation between electron transfer (ET) and proton relocations. In photosystem II membrane particles, the redox transitions of the water-oxidizing Mn complex were initiated by nanosecond laser flashes and monitored by absorption spectroscopy at 360 nm (A(360)). In the oxygen evolution transition (S(3) + hν → S(0) + O(2)), an exponential decrease in A(360) (τ(O(2)) = 1.6 ms) can be assigned to Mn reduction and O(2) formation. The corresponding rate-determining step is the ET from the Mn complex to a tyrosine radical (Y(Z)(ox)). We find that this A(360) decrease is preceded by a lag phase with a duration of 170 ± 40 μs (τ(lag) at pH 6.2), indicating formation of an intermediate before ET and O-O bond formation and corroborating results obtained by time-resolved X-ray spectroscopy. Whereas τ(O(2)) exhibits a minor kinetic isotope effect and negligible pH dependence, formation of the intermediate is slowed significantly both in D(2)O (τ(lag) increase of ～140% in D(2)O) and at low pH (τ(lag) of 30 ± 20 μs at pH 7.0 vs τ(lag) of 470 ± 80 μs at pH 5.5). These findings support the fact that in the oxygen evolution transition an intermediate is created by deprotonation and removal of a proton from the Mn complex, after Y(Z)(ox) formation but before the onset of electron transfer and O-O bond formation.     

Light-induced FTIR difference spectroscopy as a powerful tool toward understanding the molecular mechanism of photosynthetic oxygen evolution

The molecular mechanism of photosynthetic oxygen evolution remains a mystery in photosynthesis research. Although recent X-ray crystallographic studies of the photosystem II core complex at 3.0-3.5 A resolutions have revealed the structure of the oxygen-evolving center (OEC), with approximate positions of the Mn and Ca ions and the amino acid ligands, elucidation of its detailed structure and the reactions during the S-state cycle awaits further spectroscopic investigations. Light-induced Fourier transform infrared (FTIR) difference spectroscopy was first applied to the OEC in 1992 as detection of its structural changes upon the S(1)-->S(2) transition, and spectra during the S-state cycle induced by consecutive flashes were reported in 2001. These FTIR spectra provide extensive structural information on the amino acid side groups, polypeptide chains, metal core, and water molecules, which constitute the OEC and are involved in its reaction. FTIR spectroscopy is thus becoming a powerful tool in investigating the reaction mechanism of photosynthetic oxygen evolution. In this mini-review, the measurement method of light-induced FTIR spectra of OEC is introduced and the results obtained thus far using this technique are summarized.