Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

Characteristic changes of the S2/S1 difference FTIR spectrum induced by Ca2+ depletion and metal cation substitution in the photosynthetic oxygen-evolving complex

Effects of Ca2+ depletion and substitution with other metal cations on the structure of the protein matrices of the oxygen-evolving complex (OEC) and their corresponding changes upon the S1 to S2 transition were examined using Fourier transform infrared (FTIR) spectroscopy. Ca2+ depletion and further supplementation with Li+, Na+, Mg2+, Ca2+, or Sr2+ did not significantly affect the typical vibrational features in the double difference S2/S1 spectrum, including the symmetric [1365(+)/1404(-) cm(-1)] and the asymmetric [1587(+)/1566(-) cm(-1)] stretching modes of the carboxylate ligand and the amide I and II modes of the backbone polypeptides. On the other hand, supplementation with K+, Rb+, Cs+, or Ba2+ significantly modified the S2/S1 spectrum, in which the carboxylate modes disappeared and the amide I and II modes were modified. Results indicate that the binding of metal cations that have ionic radii larger than that of Ca2+ to the Ca2+ site induces perturbations in the protein matrices in the vicinity of the Mn cluster to interrupt the characteristic structural and/or conformational changes upon the oxidation of the Mn cluster accompanied with the S1 to S2 transition. The spectrum was also altered by the supplementation of Cd2+, which has an ionic radius comparable to that of Ca2+. A single-pulse-induced S2/S1 difference spectrum revealed that bands that have been assigned to the vibrational modes for the Y(Z) tyrosine and the histidine ligand for the Mn cluster were not induced in the K+-supplemented membranes, although the histidine band is likely to be preserved in the Ca2+-depleted membranes. The Y(Z) band was considerably small in the double difference S2/S1 spectrum in the Ca2+-depleted and the cation-substituted membranes but distinctively present in the Sr2+- or Ca2+-replenished membranes. Furthermore, cation supplementation induced several new bands that disappeared following the Ca2+ replenishment. These results suggest that the proper organization of the hydrogen bond network within OEC for the water oxidation chemistry requires the Ca2+ ion and indicate that the role of Ca2+ is not purely structurally defined by the physical properties of the ion, such as valence and ionic radius. On the basis of these and other findings, we propose that Ca2+ is necessary for the formation of the hydrogen bond network that is involved in the reaction step of water oxidation.     

Structural and functional modulation of the manganese cluster in Ca(2+)-depleted photosystem II induced by binding of the 24-kilodalton extrinsic protein.

Depletion of functional Ca2+ from photosystem (PS) II membranes impairs O2 evolution. Redox properties of the Mn cluster as probed by thermoluminescence were modified differently in Ca(2+)-depleted PSII depending on the procedure for Ca2+ extraction. Ca2+ depletion by low-pH treatment gave rise to an abnormally modified S2 state exhibiting a thermoluminescence band with elevated peak temperature accompanied by a marked upshift in threshold temperature for its formation, whereas Ca2+ depletion by NaCl washing in the light followed by the addition of EDTA could generate a similarly modified S2 state only when the Ca(2+)-depleted PSII was reconstituted with the 24-kDa extrinsic proteins. These results indicated that manifestation of the abnormal properties of the Ca(2+)-depleted S2 state is significantly contributed by the association of the 24-kDa extrinsic protein to PSII. It was inferred that the 24-kDa extrinsic protein regulates the structure and function of the Mn cluster in the absence of functional Ca2+ through a conformational modulation of the intrinsic protein(s) that bind(s) both Mn and Ca. Features of the extrinsic protein-dependent modulation of the Mn cluster were discussed in relation to the function of Ca2+ in O2 evolution.     

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.     

Study on the Binding State of Calcium in Photosystem ⅡOxygen-Evolution Complex

Washing spinach PSII oxygen-evolution complex (OEC) with 2 mmol/L EGTA or extraction medium caused a 28.4% and 25.0% loss of oxygen evolution activities respectively, but the loss of polypeptide components of OEC did not take place, whereas washing with 1 mol/L NaCI caused both a 90.0% loss of oxygen evolution activity and loss of 17, 23kD polypeptides. Adding 5–10 mmol/L CaC12 could restore oxygen evolution activities of OEC by various washing to a great extent, but had no effect on control OEC, whereas adding 5–10 mmol/L EGTA had no effect on the OEC by various' washing, but caused the loss of oxygen evolution mixtures, which could induce the release of of 17, 23kD polypeptides from OEC, caused 54.3% loss of oxygen evolution activity, under this circumstance, adding 2 mmol/L of EGTA could only maintain a weak oxygen evolution activity of OEC, but adding 10 mmol/L of CaCl2 could restore oxygen evolution activity of OEC to the control level. These findings' suggest a two way loose binding of Ga2+ to PSⅡ OEC in one way Ca2+ is loose bound to the surface of PSⅡOEC and in other, the Ca2+-binding site is wrapped by 17, 23kD polypeptides. Both of them have effect on oxygen evolution activity of PSⅡ OEC. By way, Mn2+ can antagonize the restoration of oxygen evolution activity by Ca2+ to the NaCl-washing PSⅡ OEC.     

X-ray spectroscopy-based structure of the Mn cluster and mechanism of photosynthetic oxygen evolution

The mechanism by which the Mn-containing oxygen evolving complex (OEC) produces oxygen from water has been of great interest for over 40 years. This review focuses on how X-ray spectroscopy has provided important information about the structure of this Mn complex and its intermediates, or S-states, in the water oxidation cycle. X-ray absorption near-edge structure spectroscopy and high-resolution Mn Kbeta X-ray emission spectroscopy experiments have identified the oxidation states of the Mn in the OEC in each of the intermediate S-states, while extended X-ray absorption fine structure experiments have shown that 2.7 A Mn-Mn di-mu-oxo and 3.3 A Mn-Mn mono-mu-oxo motifs are present in the OEC. X-ray spectroscopy has also been used to probe the two essential cofactors in the OEC, Ca2+ and Cl-, and has shown that Ca2+ is an integral component of the OEC and is proximal to Mn. In addition, dichroism studies on oriented PS II membranes have provided angular information about the Mn-Mn and Mn-Ca vectors. Based on these X-ray spectroscopy data, refined models for the structure of the OEC and a mechanism for oxygen evolution by the OEC are presented.     

Importance of the N-terminal sequence of the extrinsic 23 kDa polypeptide in Photosystem II in ion retention in oxygen evolution

The function of the extrinsic 23 kDa polypeptide (OEC23) in Photosystem II (PS II) is to retain Ca(2+) and Cl(-) during the S-state turnover of the Mn cluster in photosynthetic oxygen evolution. Recombinant OEC23s from several plant species were produced in Escherichia coli to characterize the molecular mechanism of the OEC23 function then used in reconstitution experiments. One tobacco isoform, OEC23 (2AF), had much less oxygen-evolving activity than the spinach and cucumber OEC23s when PS II activities were reconstituted in salt-washed spinach PS II particles. The fact that the OEC23s had similar binding affinities for PS II particles suggests different ion-retention capacities for the individual OEC23s: The chimeric OEC23s produced between spinach and 2AF and those produced between cucumber and 2AF show that 58 N-terminal amino acid residues are important for PS II activity. Further dissection of the sequence and site-directed mutagenesis indicated the importance of 20 N-terminal amino acid residues for activity, in particular the asparagine at the 15th position. In spinach the N15D mutation decreased PS II activity, whereas in 2AF the D15N mutation increased it. This shows the importance of the N-terminal sequence of OEC23 in ion retention during the water-splitting process.     

Structural and functional modifications of the manganese cluster in Ca(2+)-depleted S1 and S2 states: electron paramagnetic resonance and X-ray absorption spectroscopy studies.

The effect of extraction of weakly bound Ca2+ by low-pH treatment on the O2-evolving apparatus was studied by use of low-temperature electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy. In low-pH-treated PSII membranes, an S2 EPR multiline signal with modified line shape was induced by illumination at 0 degrees C, but its signal amplitude decreased upon lowering the excitation temperature with concomitant oxidation of cytochrome (cyt) b-559 in place of Mn. The half-inhibition temperature for formation of the modified multiline signal was found at -33 degrees C, which was much higher than that for formation of the normal S2 state in untreated control membranes. Signal IIf was normally induced down to -30 degrees C, but its dependence on excitation temperature was different from that for modified S2. This was interpreted as indicating that the low-temperature blockage of modified S2 formation is due to the incapability of electron abstraction from the Mn cluster. The Mn K-edge of X-ray absorption near-edge structure (XANES) spectrum shifted to lower energy by 0.8 eV after low-pH treatment, but the shift was reversed by addition of Ca2+. Upon illumination at 0 degrees C of treated membranes, the K-edge energy was up-shifted by 0.8 eV, but was not upon illumination at 210 K. These results were interpreted as indicating that extraction of weakly bound Ca2+ by low-pH treatment gives rise to structural and functional modulations of the Mn cluster.     

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.     

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.     

Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation.

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands. Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.     

Exploring the energetics of water permeation in photosystem II by multiple steered molecular dynamics simulations

The Mn(4)Ca cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the Mn(4)Ca cluster by moving through protein. Molecular dynamics simulations were used to determine the energetic barriers for water permeation though PSII extrinsic proteins. Potentials of mean force (PMFs) for water were derived by using the technique of multiple steered molecular dynamics (MSMD). Calculation of free energy profiles for water permeation allowed us to characterize previously identified water channels, and discover new pathways for water movement toward the Mn(4)Ca cluster. Our results identify the main constriction sites in these pathways which may serve as selectivity filters that restrict both the access of solutes detrimental to the water oxidation reaction and loss of Ca(2+) and Cl(-) from the active site.     

D1-Asp170 is structurally coupled to the oxygen evolving complex in photosystem II as revealed by light-induced Fourier transform infrared difference spectroscopy

We report both mid-frequency (1800-1200 cm(-)(1)) and low-frequency (670-350 cm(-)(1)) S(2)/S(1) FTIR difference spectra of photosystem II (PSII) particles isolated from wild-type and D1-D170H mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Both mid- and low-frequency S(2)/S(1) spectra of the Synechocystis wild-type PSII particles closely resemble those from spinach PSII samples, which confirms an earlier result by Noguchi and co-workers [Noguchi, T., Inoue, Y., and Tang, X.-S. (1997) Biochemistry 36, 14705-14711] and indicates that the coordination environment of the oxygen evolving complex (OEC) in Synechocystis is very similar to that in spinach. We also found that there is no appreciable difference between the mid-frequency S(2)/S(1) spectra of wild-type and of D1-D170H mutant PSII particles, from which we conclude that D1-Asp170 does not undergo a significant structural change during the S(1) to S(2) transition. This result also suggests that, if D1-Asp170 ligates Mn, it does not ligate the Mn ion that is oxidized during the S(1) to S(2) state transition. Finally, we found that a mode at 606 cm(-)(1) in the low-frequency wild-type S(2)/S(1) spectrum shifts to 612 cm(-)(1) in the D1-D170H mutant spectrum. Because this 606 cm(-)(1) mode has been previously assigned to an Mn-O-Mn cluster mode of the OEC [Chu, H.-A., Sackett, H., and Babcock, G. T. (2000) Biochemistry 39, 14371-14376], we conclude that D1-Asp170 is structurally coupled to the Mn-O-Mn cluster structure that gives rise to this band. Our results suggest that D1-Asp170 either directly ligates Mn or Ca(2+) or participates in a hydrogen bond to the Mn(4)Ca(2+) cluster. Our results demonstrate that combining FTIR difference spectroscopy with site-directed mutagenesis has the potential to provide insights into structural changes in Mn and Ca(2+) coordination environments in the different S states of the OEC.     

Binding of lanthanum and gadolinium ions to concanavalin A studied calorimetrically at 25 degrees C

The interaction between Concanavalin A (ConA) and the lanthanide ions La3+ and Gd3+ has been studied calorimetrically at 25 degrees C. The measurements were carried out at a pH of 4.5, where the protein exists prevailingly as a dimer. Calorimetry allows the direct determination of the binding enthalpy and the evaluation of both the apparent association constant, and the apparent free energy and entropy. Three groups of data were collected. The first concerns the interaction of the 'native' protein, i.e., fully metallized with Mn2+ and Ca2+, with the lanthanides. The second concerns the interaction of the completely demetallized protein with La3+ and Gd3+. Finally, the affinity of each complex was tested for the specific sugar alpha-methylmannopyranoside. The analysis of the thermodynamic parameters obtained, led to the following conclusions: 1) a specific site, named S3, exists on the protein for the lanthanides, distinct from the S1 site of the transition metal and from the S2 site, specific for calcium. There is only one S3 site per protomer when the protein has Mn2+ in S1 and Ca2+ in S2. Moreover, there is no appreciable competition for S1 and S2 from the lanthanides. The 'native' protein, metallized with La3+ or Gd3+, is a fully functional protein. 2) The demetallized protein (ApoCon A) has at least two sites per protomer for the lanthanides. The hypothesis is that, besides the S3 site, the lanthanides, in the absence of Mn2+, can also occupy the S1, but not the S2, site. The protein metallized only with gadolinium ion is completely inactive toward the interaction with the mannoside. The same happens when, along with gadolinium, only calcium or manganese is present. Hence, in the absence of the transition metal in S1 or of calcium in S2, the protein is not in the conformation suitable to interact with its specific substrate.     

The EPR signals from the S0 and S2 states of the Mn cluster in photosystem II relax differently

The oxygen evolving complex (OEC) of photosystem II (PSII) gives rise to manganese-derived electron paramagnetic resonance (EPR) signals in the S0 and S2 oxidation states. These signals exhibit different microwave power saturation behavior between 4 and 10 K. Below 8 K, the S0 state EPR signal is a faster relaxer than the S2 multiline signal, but above 8 K, the S0 signal is the slower relaxer of the two. The different temperature dependencies of the relaxation of the S0 and S2 ground-state Mn signals are due to differences in the spin-lattice relaxation process. The dominating spin-lattice relaxation mechanism is concluded to be a Raman mechanism in the S0 state, with a T(4.1) temperature dependence of the relaxation rate. It is proposed that the relaxation of the S2 state arises from a Raman mechanism as well, with a T(6.8) temperature dependence of the relaxation rate, although the data also fit an Orbach process. If both signals relax through a Raman mechanism, the different exponents are proposed to reflect structural differences in the proteins surrounding the Mn cluster between the S0 and S2 states. The saturation of SII(slow) from the Y(D)(ox) radical on the D2 protein was also studied, and found to vary between the S0 and the S2 states of the enzyme in a manner similar to the EPR signals from the OEC. Furthermore, we found that the S2 multiline signal in the second turnover of the enzyme is significantly more difficult to saturate than in the first turnover. This suggests differences in the OEC between the first and second cycles of the enzyme. The increased relaxation rate may be caused by the appearance of a relaxation enhancer, or it may be due to subtle structural changes as the OEC is brought into an active state.     

A truncated mutant of the extrinsic 23-kDa protein that absolutely requires the extrinsic 17-kDa protein for Ca2+ retention in photosystem II

One function of the extrinsic 23-kDa protein in photosystem II (OEC23) is to retain Ca(2+ )and Cl(-), two essential cofactors for photosynthetic oxygen evolution. A truncated mutant of OEC23 (OEC23 Delta19) revealed that 19 residues of the N-terminus of OEC23 were necessary for Ca(2+ )retention but not for its proper interaction with OEC17, the extrinsic 17-kDa protein in photosystem II. The lost ability of OEC23 Delta19 to reconstitute the oxygen-evolving activity was partially restored by OEC17 binding, suggesting the involvement of OEC17 in Ca(2+ )retention in photosystem II.     

Nitric oxide-induced formation of the S-2 state in the oxygen-evolving complex of photosystem II from Synechococcus elongatus

In spinach photosystem II (PSII) membranes, the tetranuclear manganese cluster of the oxygen-evolving complex (OEC) can be reduced by incubation with nitric oxide at -30 degrees C to a state which is characterized by an Mn(2)(II, III) EPR multiline signal [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. This state was recently assigned to the S(-)(2) state of the OEC [Schansker, G., Goussias, C., Petrouleas, V., and Rutherford, A. W. (2002) Biochemistry 41, 3057-3064]. On the basis of EPR spectroscopy and flash-induced oxygen evolution patterns, we show that a similar reduction process takes place in PSII samples of the thermophilic cyanobacterium Synechococcus elongatus at both -30 and 0 degrees C. An EPR multiline signal, very similar but not identical to that of the S(-)(2) state in spinach, was obtained with monomeric and dimeric PSII core complexes from S. elongatus only after incubation at -30 degrees C. The assignment of this EPR multiline signal to the S(-)(2) state is corroborated by measurements of flash-induced oxygen evolution patterns and detailed fits using extended Kok models. The small reproducible shifts of several low-field peak positions of the S(-)(2) EPR multiline signal in S. elongatus compared to spinach suggest that slight differences in the coordination geometry and/or the ligands of the manganese cluster exist between thermophilic cyanobacteria and higher plants.     

Characterization of high temperature induced stress impairments in thylakoids of rice seedlings.

Exposure of isolated thylakoids or intact plants to elevated temperature is known to inhibit photosynthesis at multiple sites. We have investigated the effect of elevated temperature (40 degrees C) for 24 hr in dark on rice seedlings to characterize the extent of damage by in vivo heat stress on photofunctions of photosystem II (PSII). Chl a fluorescence transient analysis in the intact rice leaves indicated a loss in PSII photochemistry (Fv) and an associated loss in the number of functional PSII units. Thylakoids isolated from rice seedlings exposed to mild heat stress exhibited >50% reduction in PSII catalyzed oxygen evolution activity compared to the corresponding control thylakoids. The ability of thylakoid membranes from heat exposed seedlings to photooxidize artificial PSII electron donor, DPC, subsequent to washing the thylakoids with alkaline Tris or NH2OH was also reduced by approximately 40% compared to control Tris or NH2OH washed thylakoids. This clearly indicated that besides the disruption of oxygen evolving complex (OEC) by 40 degrees C heat exposure for 24 hr, the PSII reaction centers were impaired by in vivo heat stress. The analysis of Mn and manganese stabilizing protein (MSP) contents showed no breakdown of 33 kDa extrinsic MSP and only a marginal loss in Mn. Thus, we suggest that the extent of heat induced loss of OEC must be due to disorganization of the OEC complex by in vivo heat stress. Studies with inhibitors like DCMU and atrazine clearly indicated that in vivo heat stress altered the acceptor side significantly. [14C] Atrazine binding studies clearly demonstrated that there is a significant alteration in the QB binding site on D1 as well as altered QA to QB equilibrium. Thus, our results show that the loss in PSII photochemistry by in vivo heat exposure not only alters the donor side but significantly alters the acceptor side of PSII.     

Mono-manganese mechanism of the photosystem II water splitting reaction by a unique Mn4Ca cluster

The molecular mechanism of the water oxidation reaction in photosystem II (PSII) of green plants remains a great mystery in life science. This reaction is known to take place in the oxygen evolving complex (OEC) incorporating four manganese, one calcium and one chloride cofactors, that is light-driven to cycle four intermediates, designated S(0) through S(4), to produce four protons, five electrons and lastly one molecular oxygen, for indispensable resources in biosphere. Recent advancements of X-ray crystallography models established the existence of a catalytic Mn(4)Ca cluster ligated by seven protein amino acids, but its functional structure is not yet resolved. The (18)O exchange rates of two substrate water molecules were recently measured for four S(i)-state samples (i=0-3) leading to (34)O(2) and (36)O(2) formations, revealing asymmetric substrate binding sites significantly depending on the S(i)-state. In this paper, we present a chemically complete model for the Mn(4)Ca cluster and its surrounding enzyme field, which we found out from some possible models by using the hybrid density functional theoretic geometry optimization method to confirm good agreements with the 3.0 A resolution PSII model [B. Loll, J. Kern, W. Saenger, A. Zouni , J. Biesiadka, Nature 438 (2005) 1040-1044] and the S-state dependence of (18)O exchange rates [W. Hillier and T. Wydrzynski, Phys. Chem. Chem. Phys. 6 (2004) 4882-4889]. Furthermore, we have verified that two substrate water molecules are bound to asymmetric cis-positions on the terminal Mn ion being triply bridged (mu-oxo, mu-carboxylato, and a hydrogen bond) to the Mn(3)CaO(3)(OH) core, by developing a generalized theory of (18)O exchange kinetics in OEC to obtain an experimental evidence for the cross exchange pathway from the slow to the fast exchange process. Some important experimental data will be discussed in terms of this model and its possible tautomers, to suggest that a cofactor, Cl(-) ion, may be bound to CP43-Arg357 nearby Ca(2+) ion and that D1-His337 may be used to trap a released proton only in the S(2)-state.     

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.