Structural isomers of the S2 state in photosystem II: do they exist at room temperature and are they important for function?

In nature, an oxo‐bridged Mn₄CaO₅ cluster embedded in photosystem II (PSII), a membrane‐bound multi‐subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light‐induced charge separations in the reaction center of PSII. The Mn₄CaO₅ cluster accumulates four oxidizing equivalents to enable the four‐electron four‐proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S‐states, S₀ – S₄ in the Kok cycle. One important question related to the catalytic mechanism of the oxygen‐evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S‐states and if such equilibria are essential for the mechanism of the O‐O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X‐ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S₂ state of PSII with structural data collected of the S₁, S₂ and S₃ states by serial crystallography at neutral pH (～6.5) using an X‐ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S₂ state, the room temperature crystallography data can be well‐described by just one S₂ structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.     

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) S₁-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 S₃-state; (f) XAS-based working model for the S₂–S₃ transition; (g) XAS-based working model for the S₀–S₁ 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 S₃–(S₄)–S₀ 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.     

An Intrinsically Disordered Photosystem II Subunit,PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

Photosystem II (PSII) is a membrane-bound enzyme that utilizes solar energy to catalyze the photooxidation of water. Molecular oxygen is evolved after four sequential light-driven oxidation reactions at the Mn₄CaO₅ oxygen-evolving complex, producing five sequentially oxidized states, S_n. PSII is composed of 17 membrane-spanning subunits and three extrinsic subunits, PsbP, PsbQ, and PsbO. PsbO is intrinsically disordered and plays a role in facilitation of the water oxidizing cycle. Native PsbO can be removed and substituted with recombinant PsbO, thereby restoring steady-state activity. In this report, we used reaction-induced Fourier transform infrared spectroscopy to obtain information concerning the role of PsbP, PsbQ, and PsbO during the S state cycle. Light-minus-dark difference spectra were acquired, monitoring structural changes associated with each accessible flash-induced S state transition in a highly purified plant PSII preparation (Triton X-100, octylthioglucoside). A comparison of S₂ minus S₁ spectra revealed that removal of PsbP and PsbQ had no significant effect on the data, whereas amide frequency and intensity changes were associated with PsbO removal. These data suggest that PsbO acts as an organizational template for the PSII reaction center. To identify any coupled conformational changes arising directly from PsbO, global ¹³C-PsbO isotope editing was employed. The reaction-induced Fourier transform infrared spectra of accessible S states provide evidence that PsbO spectral contributions are temperature (263 and 277 K) and S state dependent. These experiments show that PsbO undergoes catalytically relevant structural dynamics, which are coupled over long distance to hydrogen-bonding changes at the Mn₄CaO₅ cluster.     

Experimental and Theoretical Studies of Calcium Fructoborate

Calcium fructoborate samples of composition Ca(C₆H₁₀O₆BO)₂·3.5H₂O were characterized by chemical analysis, infrared and Raman spectroscopy, and thermoanalytical (thermogravimetric and differential thermal analysis) data. Theoretical studies, using density functional theory, were made for seven different structural models of the fructoborate moiety, and the most stable structure could be derived from these calculations. The results of the theoretical study also allow improving the assignment of the vibrational spectra of the compound.     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

The carboxyl-terminal processing of precursor D1 protein of the photosystem II reaction center

The D1 protein, a key subunit of photosystem II reaction center, is synthesized as a precursor form with a carboxyl-terminal extension, in oxygenic photosynthetic organisms with some exceptions. This part of the protein is removed by the action of an endopeptidase, and the proteolytic processing is indispensable for the manifestation of oxygen-evolving activity in photosynthesis. The carboxyl-terminus of mature D1 protein, which appears upon the cleavage, has recently been demonstrated to be a ligand for a manganese atom in the Mn₄Ca-cluster, which is responsible for the water oxidation chemistry in photosystem II, based on the isotope-edited Fourier transform infrared spectroscopy and the X-ray crystallography. On the other hand, the structure of a peptidase involved in the cleavage of precursor D1 protein has been resolved at a higher resolution, and the enzyme–substrate interactions have extensively been analyzed both in vivo and in vitro. The present article briefly summarizes the history of research and the present state of our knowledge on the carboxyl-terminal processing of precursor D1 protein in the photosystem II reaction center.     

Inhomogeneity of the EPR multiline signal from the S2-state of the photosystem II oxygen-evolving enzyme

The manganese complex (Mn₄) which is responsible for water oxidation in photosystem II is EPR detectable in the S₂-state, one of the five redox states of the enzyme cycle. The S₂-state is observable at 10?K either as an EPR multiline signal (spin S?=?1/2) or as a signal at g?=?4.1 (spin S?=?3/2 or 5/2). It has recently been shown that the state responsible for the multiline signal is converted to that responsible for the g?=?4.1 signal upon the absorption of near-infrared light [Boussac A, Girerd J-J, Rutherford AW (1996) Biochemistry 35?:?6984–6989]. It is shown here that the yield of the spin interconversion may be variable and depends on the photosystem II (PSII) preparations. The EPR multiline signal detected after near-infrared illumination, and which originates from PSII centers not susceptible to the near-infrared light, is shown to be different from that which originates from infrared-susceptible PSII centers. The total S₂-multiline signal results from the superposition of the two multiline signals which originate from these two PSII populations. One S₂ population gives rise to a "narrow" multiline signal characterized by strong central lines and weak outer lines. The second population gives rise to a "broad" multiline signal in which the intensity of the outer lines, at low and high field, are proportionally larger than those in the narrow multiline signal. The larger the relative amplitude of the outer lines at low and high field, the higher is the proportion of the near-infrared-susceptible PSII centers and the yield of the multiline to g?=?4.1 signal conversion. This inhomogeneity of the EPR multiline signal is briefly discussed in terms of the structural properties of the Mn₄ complex.     

Copper Toxicity Affects Photosystem II Electron Transport at the Secondary Quinone Acceptor, Q(B)

The nature of Cu²⁺ inhibition of photosystem II (PSII) photochemistry in pea (Pisum sativum L.) thylakoids was investigated monitoring Hill activity and light emission properties of photosystem II. In Cu²⁺-inhibited thylakoids, diphenyl carbazide addition does not relieve the loss of Hill activity. The maximum yield of fluorescence induction restored by hydroxylamine in Tris-inactivated thylakoids is markedly reduced by Cu²⁺. This suggests that Cu²⁺ does not act on the donor side of PSII but on the reaction center of PSII or on components beyond. Thermoluminescence and delayed luminescence studies show that charge recombination between the positively charged intermediate in water oxidation cycle (S₂) and negatively charged primary quinone acceptor of pSII (Q_A⁻) is largely unaffected by Cu²⁺. The S₂Q_B⁻ charge recombination, however, is drastically inhibited which parallels the loss of Hill activity. This indicates that Cu²⁺ inhibits photosystem II photochemistry primarily affecting the function of the secondary quinone electron acceptor, Q_B. We suggest that Cu²⁺ does not block electron flow between the primary and secondary quinone acceptor but modifies the Q_B site in such a way that it becomes unsuitable for further photosystem II photochemistry.     

Structural Changes of the Oxygen-evolving Complex in Photosystem II during the Catalytic Cycle

The oxygen-evolving complex (OEC) in the membrane-bound protein complex photosystem II (PSII) catalyzes the water oxidation reaction that takes place in oxygenic photosynthetic organisms. We investigated the structural changes of the Mn₄CaO₅ cluster in the OEC during the S state transitions using x-ray absorption spectroscopy (XAS). Overall structural changes of the Mn₄CaO₅ cluster, based on the manganese ligand and Mn-Mn distances obtained from this study, were incorporated into the geometry of the Mn₄CaO₅ cluster in the OEC obtained from a polarized XAS model and the 1.9-Å high resolution crystal structure. Additionally, we compared the S₁ state XAS of the dimeric and monomeric form of PSII from Thermosynechococcus elongatus and spinach PSII. Although the basic structures of the OEC are the same for T. elongatus PSII and spinach PSII, minor electronic structural differences that affect the manganese K-edge XAS between T. elongatus PSII and spinach PSII are found and may originate from differences in the second sphere ligand atom geometry.     

EPR spectroscopy of the manganese cluster of photosystem II

Electron paramagnetic resonance (EPR) spectroscopy is a valuable tool for understanding the oxidation state and chemical environment of the Mn₄Ca cluster of photosystem II. Since the discovery of the multiline signal from the S₂ state, EPR spectroscopy has continued to reveal details about the catalytic center of oxygen evolution. At present EPR signals from nearly all of the S-states of the Mn₄Ca cluster, as well as from modified and intermediate states, have been observed. This review article describes the various EPR signals obtained from the Mn₄Ca cluster, including the metalloradical signals due to interaction of the cluster with a nearby organic radical.     

Ozone stress‐induced proteomic changes in leaf total soluble and chloroplast proteins of soybean reveal that carbon allocation is involved in adaptation in the early developmental stage

Considerable soybean yield losses caused by ozone (O₃) stress have been demonstrated by large‐scale meta‐analyses of free‐gas concentration enrichment systems. In this study, comparative proteomic approach was employed to explore the differential changes of proteins in O₃ target structures such as leaf and chloroplasts of soybean seedlings. Acute O₃ exposure (120 parts‐per‐billion) for 3 days did not cause any visible symptoms in developing leaves. However, higher amounts of ROS and lipid peroxidation indicated that severe oxidative burst occurred. Immunoblot analysis of O₃‐induced known proteins revealed that proteins were modulated before symptoms became visible. Proteomic analysis identified a total of 20 and 32 differentially expressed proteins from O₃‐treated leaf and chloroplast, respectively. Proteins associated with photosynthesis, including photosystem I/II and carbon assimilation decreased following exposure to O₃. In contrast, proteins involved in antioxidant defense and carbon metabolism increased. The activity of enzymes involved in carbohydrate metabolism increased following exposure to O₃, which is consistent with the decrease in starch and increase in sucrose concentrations. Taken together, these results suggest that carbon allocation is tightly programmed, and starch degradation probably feeds the tricarboxylic acid cycle while the photosynthesis pathway is severely affected during O₃ stress.     

Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

Oxygen ligand exchange at metal sites – implications for the O2 evolving mechanism of photosystem II

The mechanism for photosynthetic O₂ evolution by photosystem II is currently a topic of intense debate. Important questions remain as to what is the nature of the binding sites for the substrate water and how does the O–O bond form. Recent measurements of the ¹⁸O exchange between the solvent water and the photogenerated O₂ as a function of the S-state cycle have provided some surprising insights to these questions (W. Hillier, T. Wydrzynski, Biochemistry 39 (2000) 4399–4405). The results show that one substrate water molecule is bound at the beginning of the catalytic sequence, in the S₀ state, while the second substrate water molecule binds in the S₃ state or possibly earlier. It may be that the second substrate water molecule only enters the catalytic sequence following the formation of the S₃ state. Most importantly, comparison of the observed exchange rates with oxygen ligand exchange in various metal complexes reveal that the two substrate water molecules are most likely bound to separate Mn^III ions, which do not undergo metal-centered oxidations through to the S₃ state. The implication of this analysis is that in the S₁ state, all four Mn ions are in the +3 oxidation state. This minireview summarizes the arguments for this proposal.     

Phenyl Disulfide Additive for Solution‐Mediated Carbon Dioxide Utilization in Li–CO2 Batteries

Phenyl disulfide (PDS) is employed as an electrolyte additive in lithium–carbon dioxide (Li–CO₂) batteries to allow for a solution‐mediated carbon dioxide reduction pathway. Thiophenolate anions, generated via electrochemical reduction of PDS, act as CO₂ capture agents by forming the adduct S‐phenyl carbonothioate (SPC^?) in solution. A mechanism of SPC^?‐mediated CO₂ capture and utilization is proposed and supported via carbon‐13 nuclear magnetic resonance spectroscopy and Fourier‐transform infrared spectroscopy. Reversible formation and decomposition of lithium carbonate and amorphous carbon during cycling, facilitated by the solution‐mediated pathway, are demonstrated with an array of characterization techniques. Li–CO₂ batteries employing the PDS additive show vastly improved capacity, energy efficiency, and cycle life. The enhanced Li–CO₂ battery performance offered by the proposed solution‐mediated reaction pathway offers a compelling step forward in the pursuit of reversible CO₂ utilization.     

Perturbations at the chloride site during the photosynthetic oxygen-evolving cycle

Photosystem II (PSII) catalyzes the oxidation of water to O₂ at the manganese-containing, oxygen-evolving complex (OEC). Photoexcitation of PSII results in the oxidation of the OEC; four sequential oxidation reactions are required for the generation and release of molecular oxygen. Therefore, with flash illumination, the OEC cycles among five S _n states. Chloride depletion inhibits O₂ evolution. However, the binding site of chloride in the OEC is not known, and the role of chloride in oxygen evolution has not as yet been elucidated. We have employed reaction-induced FT-IR spectroscopy and selective flash excitation, which cycles PSII samples through the S state transitions. On the time scale employed, these FT-IR difference spectra reflect long-lived structural changes in the OEC. Bromide substitution supports oxygen evolution and was used to identify vibrational bands arising from structural changes at the chloride-binding site. Contributions to the vibrational spectrum from bromide-sensitive bands were observed on each flash. Sulfate treatment led to an elimination of oxygen evolution activity and of the FT-IR spectra assigned to the S₃ to S₀ (third flash) and S₀ to S₁ transitions (fourth flash). However, sulfate treatment changed, but did not eliminate, the FT-IR spectra obtained with the first and second flashes. Solvent isotope exchange in chloride-exchanged samples suggests flash-dependent structural changes, which alter protein dynamics during the S state cycle. Supported by NSF MCB 03-55421.     

Spectroscopic exploration and thermodynamic characterization of desvenlafaxine interacting with fluorescent bovine serum albumin

The mechanism of the interaction between bovine serum albumin (BSA) and desvenlafaxine was studied using fluorescence, ultraviolet absorption, 3‐dimensional fluorescence spectroscopy, circular dichroism, synchronous fluorescence spectroscopy, cyclic voltametry, differential scanning calorimetry, and attenuated total reflection–Fourier transform infrared spectroscopic techniques under physiological condition at pH 7.4. Stern‐Volmer calculations authenticate the fluorescence of BSA that was quenched by desvenlafaxine in a collision quenching mode. The fluorescence quenching method was used to evaluate number of binding sites “n” and binding constant K _A that were measured, and various thermodynamic parameters were evaluated at different temperatures by using the van't Hoff equation and differential scanning calorimetry technique, which indicated a spontaneous and hydrophobic interaction between BSA and desvenlafaxine. According to the Förster theory we calculate the distance between the donor, BSA and acceptor, desvenlafaxine molecules. Furthermore, circular dichroism and attenuated total reflection–Fourier transform infrared spectroscopy indicate nominal changes in the secondary structure of the protein.     

Photochemical Apparatus Organization in the Chloroplasts of Two Beta vulgaris Genotypes

The composition and structural organization of thylakoid membranes of a low chlorophyll mutant of Beta vulgaris was investigated using spectroscopic, kinetic and electrophoretic techniques. The data obtained were compared with those of a standard F1 hybrid of the same species. The mutant was depleted in chlorophyll b relative to the hybrid and it had a higher photosystem II/photosystem I reaction center (Q/P₇₀₀) ratio and a smaller functional chlorophyll antenna size. Analysis of thylakoid membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the mutant lacked a portion of the chlorophyll a/b light-harvesting complex but was enriched in the photosystem II reaction center chlorophyll protein complex. Comparison of functional antenna sizes and of photosystem stoichiometries determined electrophoretically were in good agreement with those determined spectroscopically. Both approaches indicated that about 30% of the total chlorophyll was associated with photosystem I and about 70% with photosystem II. A greater proportion of photosystem II_β was detected in the mutant. The results suggest that a higher photosystem II to photosystem I ratio in the sugar beet mutant has apparently compensated for the smaller photosystem II chlorophyll light-harvesting antenna in its chloroplasts. Moreover, a lack of chlorophyll a/b light-harvesting complex correlates with the abundance of photosystem II_β. It is proposed that a developmental relationship exists between the two types of photosystem II where photosystem II_β is a precursor form of photosystem II_α occurring prior to the addition of the chlorophyll a/b light-harvesting complex and grana formation.     

Herbicide binding and thermal stability of photosystem II isolated from Thermosynechococcus elongatus

Binding of herbicides to photosystem II inhibits the electron transfer from Q_A to Q_B due to competition of herbicides with plastoquinone bound at the Q_B site. We investigated herbicide binding to monomeric and dimeric photosystem II core complexes (PSIIcc) isolated from Thermosynechococcus elongatus by a combination of different methods (isothermal titration and differential scanning calorimetry, CD spectroscopy and measurements of the oxygen evolution) yielding binding constants, enthalpies and stoichiometries for various herbicides as well as information regarding stabilization/destabilization of the complex. Herbicide binding to detergent-solubilized PSIIcc can be described by a model of single independent binding sites present on this important membrane protein. Interestingly, binding stoichiometries herbicide:PSIIcc are lower than 1:1 and vary depending on the herbicide under study. Strong binding herbicides such as terbutryn stabilize PSIIcc in thermal unfolding experiments and endothermically binding herbicides like ioxynil probably cause large structural changes accompanied with the binding process as shown by differential scanning calorimetry experiments of the unfolding reaction of PSIIcc monomer in the presence of ioxynil. In addition we studied the occupancy of the Q_B sites with plastoquinone (PQ9) by measuring flash induced fluorescence relaxation yielding a possible explanation for the deviations of herbicide binding from a 1:1 herbicide/binding site model.     

Secondary structure and dynamics study of the intrinsically disordered silica‐mineralizing peptide P5S3 during silicic acid condensation and silica decondensation

The silica forming repeat R5 of sil1 from Cylindrotheca fusiformis was the blueprint for the design of P₅S₃, a 50‐residue peptide which can be produced in large amounts by recombinant bacterial expression. It contains 5 protein kinase A target sites and is highly cationic due to 10 lysine and 10 arginine residues. In the presence of supersaturated orthosilicic acid P₅S₃ enhances silica‐formation whereas it retards the dissolution of amorphous silica (SiO₂) at globally undersaturated concentrations. The secondary structure of P₅S₃ during these 2 processes was studied by circular dichroism (CD) spectroscopy, complemented by nuclear magnetic resonance (NMR) spectroscopy of the peptide in the absence of silicate. The NMR studies of dual‐labeled (¹³C, ¹⁵N) P₅S₃ revealed a disordered structure at pH 2.8 and 4.5. Within the pH range of 4.5‐9.5 in the absence of silicic acid, the CD data showed a disordered structure with the suggestion of some polyproline II character. Upon silicic acid polymerization and during dissolution of preformed silica, the CD spectrum of P₅S₃ indicated partial transition into an α‐helical conformation which was transient during silica‐dissolution. The secondary structural changes observed for P₅S₃ correlate with the presence of oligomeric/polymeric silicic acid, presumably due to P₅S₃‐silica interactions. These P₅S₃‐silica interactions appear, at least in part, ionic in nature since negatively charged dodecylsulfate caused similar perturbations to the P₅S₃ CD spectrum as observed with silica, while uncharged ß‐d ‐dodecyl maltoside did not affect the CD spectrum of P₅S₃. Thus, with an associated increase in α‐helical character, P₅S₃ influences both the condensation of silicic acid into silica and its decondensation back to silicic acid.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S₁-to-S₂ transition was obtained with Ca²⁺-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca²⁺ to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S₂/S₁ FTIR spectrum upon Ca²⁺ depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca²⁺ depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K⁺ at the Ca²⁺ site. In the present study, the preparation of the Ca²⁺-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K⁺. The obtained S₂/S₁ spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm^− 1 and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K⁺. This result indicates that the drastic FTIR changes are a pure effect of Ca²⁺ depletion, and provides solid evidence for the general view that Ca²⁺ is strongly coupled with the Mn cluster.