Water-oxidizing Complex Research Articles

Mechanistic and structural aspects of photosynthetic water oxidation

Conclusions on the functional and structural organisation of photosynthetic water oxidation are gathered from a critical survey of the wealth of data reported in the literature and author's own experimental research: (1) the water oxidising complex (WOC) contains a tetranuclear manganese cluster of dimer of dimers' structure and functional heterogeneity of the metal centers, (2) the four step univalent oxidative pathway leading to water oxidation into molecular oxygen and four protons comprises only manganese, tyrosine YZ of polypeptide Dl and the substrate as redox active species, (3) the redox transitions S0→ S1 and S1→ S2 are manganese centered whereas S2→ S3 is most likely a ligand‐centered reaction, (4) there exist several lines of evidence for a marked structural change that accompanies the redox transition S2→ S3, (5) one Ca2+ is an indispensible constituent of a functionally competent WOC while the role of Cl is much less clear and a direct participation disputable, (6) substrate water is most likely bound in all redox states S0,…,S3 and exchangeable with the bulk phase. The protonation state is determined by the redox state S1 and the protein microenvironment. A mechanism is proposed for water oxidation in the WOC that is based on three key postulates: (1) water oxidation takes place in the first coordination sphere of one manganese dimer [MnaMnb]; (2) the essential O‐O bond is preformed in S3 as part of a rapid redox isomerism S3(I)→S3(II) where in S3(II) a nuclear geometry and electronic configuration is attained that corresponds to a peroxidic‐type species; and (3) S3(II) is an ‘entatic state’ for the formation of complexed dioxygen triggered by YZOX induced electron abstraction from the WOC and electronic redistribution to S0(O2).

Analysis of the reaction coordinate of photosynthetic water oxidation by kinetic measurements of 355 nm absorption changes at different temperatures in photosystem II preparations suspended in either H2O or D2O.

Flash-induced absorption changes at 355 nm were measured at different temperatures within the range of 2 degrees C </= theta </= 25 degrees C in dark-adapted PS II core complexes from spinach [O2 evolution rate: 1500 +/- 100 micromol of O2 (mg of Chl)-1 h-1] that were dissolved either in H2O- or in D2O-containing buffer. Comparative measurements were performed at 20 degrees C in H2O- or D2O-containing suspensions of PS II membrane fragments [O2 evolution rate: 600 +/- 40 micromol of O2 (mg of Chl)-1 h-1]. The results obtained reveal the following: (a) The activation energies of the individual redox steps in the water oxidizing complex (WOC) are dependent on the redox state Si with EA(S1-->S2) = 14 kJ/mol, EA(S2-->S3) = 35 kJ/mol, and EA(S3-->-->S0 + O2) = 21 kJ/mol for theta > 11 degrees C, 67 kJ/mol for theta < 11 degrees C in PS II core complexes dissolved in H2O; (b) replacement of exchangeable protons by deuterons causes only minor changes (</=15%) of the activation energies; and (c) the rate constants of these reactions in PS II core complexes are characterized by H/D isotope ratios, ki(H)/ki(D), of 1.6, 2.3, and 1.5 for the transitions S1 --> S2, S2 --> S3, and S3 -->--> S0 + O2, respectively. The corresponding values of PS II membrane fragments are 1.3, 1.3, and 1. 4. Based on these results and corresponding EA data reported in the literature for PS II membrane fragments from spinach [Renger, G., & Hanssum, B. (1992) FEBS Lett. 299, 28-32] and PS II particles from the thermophilic cyanobacterium Synechococcus vulcanus Copeland [Koike, H., Hanssum, B., Inoue, Y., & Renger, G. (1987) Biochim. Biophys. Acta 893, 524-533], the reaction coordinate of the redox sequence in the WOC is inferred to be almost invariant to the evolutionary development from cyanobacteria to higher plants. Furthermore, the rather high activation energy of the S2 --> S3 transition provides evidence for a significant structural change coupled with this reaction. Implications for the mechanism of photosynthetic water oxidation are discussed.

Quantitative Kinetic Model for Photoassembly of the Photosynthetic Water Oxidase from Its Inorganic Constituents: Requirements for Manganese and Calcium in the Kinetically Resolved Steps,

The process of photoactivation, the assembly of a functional water-oxidizing complex (WOC) from the apoproteins of photosystem II of higher plants and inorganic cofactors (Mn2+, Ca2+, and Cl-), was known from earlier works to be a two-step kinetic process, requiring two light-induced processes separated by a slower dark period. However, these steps had not been directly resolved in any kinetic experiment, until development of an ultrasensitive polarographic O2 electrode and synthesis of an improved chelator for cofactor removal allowed direct kinetic resolution of the first pre-steady state intermediate [Ananyev, G. M. & Dismukes, G. C. (1996a) Biochemistry 35, 4102-4109]. Herein, the dependence of the rates of each of the first two light steps and the dark step of photoactivation was directly determined in spinach PSII membranes over a range of calcium and manganese concentrations at least 10-fold lower than those possible using commercial O2 electrodes. The following results were obtained. (1) One Mn2+ ion binds and is photooxidized to Mn3+ at a high-affinity site, forming the first light-induced intermediate, IM1. Formation of IM1 is coupled to the dissociation of a bound Ca2+ ion either located in the Mn site or coupled to it. (2) The inhibition constant for Ca2+ dissociation from this site is equal to 1.5 mM. (3) The dissociation constant of Mn2+ at this high-affinity site is equal to 8 microM at the optimum calcium concentration for O2-evolving activity of 8 mM, in agreement with the high-affinity site for electron donation to PSII. (4) Prior to the next photolytic step, one Ca2+ ion must bind at its effector site so that stable photooxidation of a second Mn2+ ion can occur, forming the second light-induced intermediate, IM2. This dark process is the rate-determining step. (5) The Michaelis constant for recovery of O2 evolution by Ca2+ binding at this effector site (Km) is equal to 1.4 mM, a value that is the same as that measured for the calcium requirement for O2 evolution in intact PSII. (6) The low quantum yield for the formation of IM2 from IM1 increases linearly with the duration of the dark period up to the longest period we could examine (10 s). Accordingly, the rate limitation in the second photolytic step originates from a slow calcium-induced dark rearrangement of the first intermediate, IM1, which we propose to be a protein conformational change that allows stable binding of the next Mn2+ ion. We further propose that the single Ca2+ ion which is required for assembly of the Mn4 cluster is equivalent to the Ca2+ ion which functions at the "gatekeeper" site in intact O2-evolving centers, where it plays a role in limiting substrate access to the Mn4 cluster [Sivaraja, M., et al. (1989) Biochemistry 28, 9459-9464; Tso, J., et al., (1991) Biochemistry 30, 4734-4739]. A molecular model for photoactivation is proposed and discussed.

Low-temperature interactions of NO with the S1 and S2 states of the water-oxidizing complex of photosystem II. A novel Mn-multiline EPR signal derived from the S1 state.

The spin-1/2-carrying NO molecule interacts with both the S1 and S2 states of the water oxidizing complex. The intermediates of the interaction can be resolved and trapped by NO treatment at subzero temperatures. At -30 degrees C and in the presence of approx. 500-700 microM NO, S1 loses the ability to yield by illumination an EPR active S2-state with an approximate half-time of 40-60 min. At longer incubation times (t1/2 = 4-5 h), an intense new multiline signal develops. The new signal has a hyperfine splitting similar to the S2 multiline [Dismukes, G. C., & Siderer, Y. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 274-278], but a modified shape with intense lines on the high field side. The NO modified S1 state can act as a low-temperature electron donor yielding an EPR silent state upon illumination at 200 K. NO interacts also with the S2 state of the water oxidizing complex rapidly at temperatures as low as -75 degrees C, to yield an EPR silent state. The rates of the latter interaction show analogies to the ammonia binding to the S2 state. It is possible, however, that NO, unlike ammonia, destabilizes the S2 state. On the basis of preliminary experiments with varying chloride concentrations in the range 0.1-50 mM, the S1 multiline state is attributed to binding of NO at a chloride sensitive site on the Mn cluster. The rapid interactions with the S2 state as well as the intermediate binding to the S1 state are less well understood at present, but they are tentatively assigned to the chloride-insensitive site of ammonia binding in the Mn cluster.

EPR investigation of water oxidizing photosystem II: detection of new EPR signals at cryogenic temperatures.

Experiments are described which allow the detection and characterization of new EPR signals in photosystem II (PSII). PSII has been extensively studied with the water oxidising complex (WOC) poised in the S1 and S2 states. Other stages in the cycle of water oxidation lack characteristic EPR signals for use as probes. In this study, experiments use multiple turnovers of PSII from an initial S1 state to allow new states of PSII to be studied. The first EPR signal detected, centered at g = 4.85 and termed the g = 5 signal, is suggested to be a new form of S2 probably formed by decay of S3 at cryogenic temperatures, but a novel form of oxidized non-heme iron cannot be fully excluded at present. The second signal is split around g = 2 and shows characteristics of signals formed by spin-spin interaction between two paramagnetic species. The split g = 2 signal is reversibly formed by illumination at <30 K of a sample containing the g = 5 signal. The g = 2 signal may be a form of the "S3" EPR signal previously only found in a variety of PSII preparations where oxygen evolution has been inhibited. Those "S3" signals are thought to arise from the interaction of an oxidized amino acid radical and the S2 state, i.e., S2X+. Illumination at higher temperatures or illumination at <30 K, followed by dark-adaptation at 77 K, removes the g = 5 signal and prevents subsequent detection of the g = 2 signal on illumination at <30 K. The most likely explanation of our data is that illumination at <30 K of centers containing the g = 5 species allows accumulation of an oxidized intermediate and that at higher temperatures electron transfer proceeds to re-form an EPR-silent S state equivalent to that initially trapped during sample preparation. Study of these signals should provide an important new insight into the WOC and PSII.

Photoinhibition of photosynthesis without net loss of photosystem II components inPopulus deltoides

The photoinhibition of photosynthesis was investigated on intact attached leaves and isolated thylakoid membranes ofPopulus deltoides.Our studies demonstrate that in intact leaves photoinhibition takes place under high irradiance which is more pronounced at higher temperatures. No net loss of Dl and other proteins associated with photosystem II (PSII) were observed even after 64 % photoinhibition suggesting that the degradation of polypeptides associated with PSII is not the only key step responsible for photoinhibition as observed by other workers. Electron transport studies in isolated thylakoid membranes suggested water oxidation complex as one of the damaged site during high light exposure. The possible mechanisms of photoinhibition without net loss of D1 protein are discussed.

Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem II.

It is shown that restoration of photoinduced electron flow and O2 evolution with Mn2+ in Mn-depleted photosystem II (PSII) membrane fragments isolated from spinach chloroplasts is considerably increased with bicarbonate in the region pH 5.0-8.0 in bicarbonate-depleted medium. In buffered solutions equilibrated with the atmosphere (nondepleted of bicarbonate), the bicarbonate effect is observed only at pH lower than the pK of H2CO3 dissociation (6.4), which indicates that HCO3- is the essential species for the restoration effect. The addition of just 2 Mn2+ atoms per one PSII reaction center is enough for the maximal reactivation when bicarbonate is present in the medium. Analysis of bicarbonate concentration dependence of the restoration effect reveals two binding sites for bicarbonate with apparent dissociation constant (Kd) of approximately 2.5 microM and 20-34 microM when 2,6-dichloro-p-benzoquinone is used as electron acceptor, while in the presence of silicomolybdate only the latter one remains. Similar bicarbonate concentration dependence of O2 evolution was obtained in untreated Mn-containing PSII membrane fragments. It is suggested that the Kd of 20-34 microM is associated with the donor side of PSII while the location of the lower Kd binding site is not quite clear. The conclusion is made that bicarbonate is an essential constituent of the water-oxidizing complex of PSII, important for its assembly and maintenance in the functionally active state.

Analysis of pH-induced structural changes of the isolated extrinsic 33 kilodalton protein of photosystem II.

Structural properties of the isolated extrinsic regulatory 33 kDa protein of the water-oxidizing complex were analyzed at different pH values. It was found that (a) titrations of the buffer capacity reveal a characteristic hysteresis effect that is unique for the 33 kDa subunit and is not observed for the other extrinsic proteins, (b) changes of the emission from the fluorescence probe 1,8-ANS are indicative of an increased accessibility of the hydrophobic core of the 33 kDa protein to the dye at lower pH, (c) the near-UV circular dichroism spectrum of the polypeptide is altered owing to a pH decrease from 6.8 to 3.8 and becomes drastically changed at pH 2.8, and (d) the content of secondary structure elements remains virtually constant in the range 3.8 < pH < 6.8, with the following values gathered from far-UV CD spectra: approximately 8% alpha-helix, approximately 33% beta-strand, approximately 15% turns, and approximately 44% random coil. Further acidification down to pH 2.8 gives rise to a decreased alpha-helix and increased beta-strand and random coil content. A theoretical model [Ptitsyn, O., & Finkelstein, A. (1983) Biopolymers 2, 15-22] was used to predict the probability and location of secondary structure elements within the protein sequence. On the basis of these calculations, an extended hydrophobic beta-sheet domain could exist in the center of the protein and an alpha-helix in the C-terminal region. From these data, the 33 kDa protein is inferred to change its tertiary structure in vitro upon acidification of the aqueous environment. Possible implications of these features are discussed.

Thermoluminescence studies on the photoinhibition of pothos leaf discs at chilling, room and high temperature

Photoinhibition in pothos leaf discs was studied by measurement of thermoluminescence inhibition and oxygen evolution. At chilling and high temperature (4 and 40 °C respectively), photoinhibition is at least 40%–50% greater than that at room temperature (25°C), suggesting greater protection from inhibition at room temperature. Flash excitation studies exhibit thermoluminescence oscillations in 50% photoinhibited and normal leaf discs, with the same periodicity of four, with maxima at the second and sixth flashes, suggesting normal functioning of the water oxidation complex in these samples. Of the four distinct thermoluminescence bands observed in pothos leaf discs, peak V (C band) is hardly photoinhibited even when the other bands show more than 80% inhibition. The lack of inhibition of peak V was further confirmed in leaf materials photoinhibited at different temperatures, indicating that this band is indeed resistant to photoinhibition. Oxygen evolution measurements in chloroplasts isolated from leaf discs photoinhibited for 30 and 60 min at 4, 25 and 40°C using dichlorobenzoquinone or ferricyanide as electron acceptor exhibit relatively higher inhibition with ferricyanide as acceptor. On the basis of these results, it is suggested that leaves under stress conditions show enhanced photoinhibition. The acceptor side of photosynthetic electron transport seems to be more susceptible to photoinhibition and the charge pair responsible for generating peak V appears to be insensitive to photoinhibition.

Kinetics of S2 and S3 reduction by tyrosine Y(D) and other endogenous donors as a function of temperature in spinach PS II membrane fragments with a reconstituted plastoquinone pool.

The characteristic period four oscillation patterns of oxygen evolution induced by a train of single-turnover flashes were measured as a function of temperature in dark-adapted photosystem II (PS II) membrane fragments that were reconstituted with native plastoquinone-9 (PQ-9) by a recently developed procedure [Kurreck, J., Seeliger, A. G., Reifarth, F., Karge, M., & Renger, G. (1995) Biochemistry 34, 15721-15731]. The following results were obtained: (a) within the range 0-35 degrees C, the probabilities of misses (alpha) and double-hits (beta) and the dark population of redox state S1 exhibit similar dependencies on the temperature; (b) below a characteristic temperature theta(c) these parameters remain virtually independent of temperature, above theta(c) (theta(c) = 20 degrees C for alpha and beta; theta(c) = 30 degrees C for S1) the values of alpha and beta increase whereas S1 decreases; and (c) the dark decay of S2 and S3 via fast and slow kinetics owing to reduction of the water oxidase by Y(D) and other endogenous electron donor(s), respectively, exhibits comparatively strong temperature dependencies with the following activation energies: E(A)(S2(fast)) = 60 +/- 10 kJ/mol, EA(S3(fast)) = 55 +/- 10 kJ/mol, E(A)(S2(slow)) = 80 +/- 5 kJ/mol, and E(A)(S3(slow)) = 75 +/- 5 kJ/mol. These values of PQ-9 reconstituted PS II membrane fragments are very similar to those that were previously reported for thylakoids [Messinger, J., Schröder, W. P., & Renger, G. (1993) Biochemistry 32, 7658-7668]. These findings reveal that the reaction coordinates of feeding electrons by endogenous electron donors into the water oxidizing complex (WOC) that attains the redox states S2 and S3 is virtually invariant to Triton X-100 treatment used in the isolation procedure of PS II membrane fragments from thylakoids. Implications of these findings are discussed.

Synthetic Water-Oxidation Catalysts for Artificial Photosynthetic Water Oxidation.

This review focuses primarily on homogeneous catalysts for the oxidation of water, but does include selected heterogeneous systems. It does not attempt to summarize all chemistry related to systems that are capable of oxidizing water. In particular, heterogeneous catalysts that have no direct relevance to understanding the WOC [water-oxidizing complex] are not discussed in detail. Neither are nonbiomimetic systems for artificial photosynthesis or water-splitting in its elements discussed. However, a list of some of the recent references in these areas can be found in section X (Nonbiomimetic Water Oxidation Catalysts). The authors will first give a brief summary of the current view of the photosynthetic WOC and its functionality, followed by analysis of the thermodynamic and kinetic constraints for water-oxidation that have to be overcome by any catalyst. Since manganese is the metal that performs this reaction in the WOC, manganese catalysts will be discussed first, followed by other transition metals, particularly ruthenium. In the final section the authors summarize the principles of reactivity learned from theory and existing models that will guide one toward synthesis of better catalysts in the future. This review does not attempt to summarize manganese chemistry relevant to nonfunctional (structural) models of the WOC, whichmore » has been reviewed recently. Water-oxidation catalysts are intrinsically important in their own right, independent of possible biological relevance. They have direct applications as catalysts in artificial photosynthetic systems for the splitting of water that could be used in future fuel cells for the generation of electricity. 133 refs.« less

Involvement of Carboxyl Groups of the PSII Reaction Center Proteins in Photoactivation of the Apo-Water-Oxidizing Complex

Involvement of residues of acidic amino acids in photoligation of manganese into the apo-water-oxidizing complex was investigated by use of l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), a water-soluble carboxyl modifier. Treatment of Mn-depleted PSII membranes by EDC in the presence of nucleophiles induced a loss of photoactivation capability in the Mn complex and partial loss of capability of photooxidation of Mil 2 *, but little decrease in the DCIP photoreduction supported by diphenylcarbazide. The inhibition of diphenylcarbazide-photooxidation by submicromolar Mn 2+ , indicative of the intactness of high-affinity Mn-binding sites, was apparently abolished by EDC treatment. From amino acid quantitation analysis of Dl and D2 proteins and CP47 of the chemically-modified membranes, approximately three carboxyl groups of the Dl protein were found to be chemically-modified with EDC after removal of the functional Mn. These results suggest that acidic amino acids on the Dl protein are involved in photoactivation of the apo-water-oxidizing complex and probably in ligation of Mn to the water-oxidizing complex.

Influence of KF, DCMU and removal of Ca + on the high-spin EPR signal of the cytochrome b-559 heme Fe(III) ligated by OH − in chloroplasts

An EPR signal at g = 6.8 attributed to the cytochrome (Cyt) b-559 heme Fe(III) ligated by OH − (Fiege, R., Schrieber, U., Lubitz, W., Renger, G. and Shuvalov, V.A. (1995) FEBS Lett. 377, 325–329) was studied. This signal is observed in intact chloroplasts when oxidized by 10 mM 2,3-dicyano,5,6-dichloro- p-benzoquinone (DDQ), but not when 5 mM p-benzoquinone is added. Addition of KF (100 mM) or removal of Ca 21 for blocking the water-oxidizing complex considerably decreases the heme Fe(III)-OH − EPR signal. In contrast, DCMU does not decrease this signal and does not influence its photochemical changes at 140 K. Thus, the EPR spectrum of Cyt b-559 Fe(III) ligated by OH − is not affected by changes at the acceptor side of Photosystem 11, and its photochemical decrease is probably not due to reduction via the acceptor side. Comparison of the effect of KF on the model heme Fe(III) in myoglobin (Mb) at pH 10.5 shows that F − replaces OH − as a ligand at the sixth coordination position of the heme Fe(III) in both Mb and chloroplasts Cyt b-559. This replacement is accompanied by changes of the symmetry of the molecular field causing a disappearance of the EPR signals at g = 6.8 and 5.0. Our results provide further evidence for a possible participation of the Cyt b-559 heme Fe ligated by OH − in photosynthetic water oxidation.

Thermoluminescence from the photosynthetic apparatus

One of the fundamental discoveries of W. Arnold was the detection of thermally stimulated light emission from preilluminated photosynthetic material (Arnold and Sherwood (1957) Proc Natl Acad Sci USA 43: 105-114). This phenomenon, called thermoluminescence (TL), is characteristic of a wide range of materials (semiconductors, minerals, inorganic and organic crystals, and complex biological systems such as the photosynthetic apparatus) which share the common ability of storing radiant energy in thermally stabilized trap states.The original discovery of TL in dried chloroplasts later proved to be a phenomenon common to all photosynthetic organisms: photosynthetic bacteria, cyanobacteria, algae and higher plants. Following the pioneering work of Arnold, considerable effort has been devoted to identification and characterization of photosynthetic TL components. This work has firmly established the participation of various redox states of the water-oxidizing complex and the quinone electron acceptors of Photosystem II in the generation of photosynthetic glow curves. Since TL characteristics are very sensitive to subtle changes in redox properties of the involved electron transport components, the TL method has become a powerful tool in probing a wide range of PS II redox reactions. In this paper, we will review the impact of Arnold's work in initiating and promoting TL studies in photosynthesis and will cover the most important developments of this field of research until the present day.

Thermoluminescence studies on the function of Photosystem II in the desiccation tolerant lichen Cladonia convoluta

The effect of desiccation and rehydration on the function of Photosystem II has been studied in the desiccation tolerant lichen Cladonia convoluta by thermoluminescence. We have shown that in functional fully hydrated thalli thermoluminescence signals can be observed from the recombination of the S2(3)QB (-) (B band), S2QA (-) (Q band), Tyr-D(+)QA (-) (C band) and Tyr-Z(+)(His(+))QA (-) (A band) charge stabilization states. These thermoluminescence signals are completely absent in desiccated thalli, but rapidly reappear on rehydration. Flash-induced oscillation in the amplitude of the thermoluminescence band from the S2(3)QB (-) recombination shows the usual pattern with maxima after 2 and 6 flashes when rehydration takes place in light. However, after rehydration in complete darkness, there is no thermoluminescence emission after the 1 st flash, and the maxima of the subsequent oscillation are shifted to the 3rd and 7th flashes. It is concluded that desiccation of Cladonia convoluta converts PS II into a nonfunctional state. This state is characterized by the lack of stable charge separation and recombination, as well as by a one-electron reduction of the water-oxidizing complex. Restoration of PS II function during rehydration can proceed both in the light and in darkness. After rehydration in the dark, the first charge separation act is utilized in restoring the usual oxidation state of the water-oxidizing comples.

Effects of hydrogen/deuterium exchange on photosynthetic water cleavage in PS II core complexes from spinach

H/D isotope exchange effects on P680 +· reduction by Y z and electron abstraction from the water oxidizing complex (WOC) in redox state S 3 by Y Z OX were analyzed in PS II core complexes from spinach by measurements of laser flash induced absorption changes at 820 nm and 355 nm. The results obtained reveal: (1) the rate of Y Z oxidation by P680 +· is almost independent of the substitution of exchangeable protons by deuterons; and (2) the reaction between Y Z OX and the WOC in S 3 exhibits a kinetic H/D isotope exchange effect of similar magnitude as that recently observed in PS II membrane fragments [Renger, G., Bittner, T. and Messinger, J. (1994) Biochem. Soc. Trans. 22, 318–322]. Based on these results it is inferred that photosynthetic dioxygen formation comprises the cleavage of at least one hydrogen bond.

Induction of luminol chemiluminescence by the manganese cluster of the photosystem II water-oxidizing complex to the S0, S1, S2, and S3 states.

Luminol chemiluminescence (CL) (lambda(max) = 425 nm) induced by the manganese cluster of photosystem II (PSII) (t(max) = 1-5 min) along with CL induced by mono-, di-, or tetranuclear manganese coordination complexes (t(max) = 2-40 s) is observed when either 200 mM sodium phosphate or 1 M Tris-HCl + 200 microM EDTA are present in the reaction medium (pH 8.5) containing peroxidase. This light emission is not observed when Tris is used in the reaction medium without EDTA. The yield of a given CL without peroxidase is 5%-20% of that with peroxidase. The peroxidase-dependent CL is inhibited by catalase (I50 = 0.14-0.17 mu M), while the peroxidase-independent CL is not inhibited by 100 mM ethanol, 1 mM NaN3, 20 mu M Cyt c, or 0.6 mu M catalase. The CL induced by the Mn cluster of the water-oxidizing complex (WOC) in the S3 or S2 state exceeds that of lower S states by 15-20-fold. The magnitude of CL induced by Mn complexes is dependent on the ligand type of the complex. The ligand types of the Mn complexes and the WOC in different S states are ranked according to the magnitude of the induced CL: 1,10-phenanthroline > 2.2'-bipyridine > WOC (S3 or S2) > hydrotris(pirazolyl) borate > WOC (S0-2 or S0, S1) > 1,4,7-triazacyclononane. It is concluded that CL is caused by H2O2 formed as a result of the oxidation of luminol by triplet molecular oxygen. The Mn cluster of WOC and manganese coordination complexes, acting as catalysts of this reaction, show oxidase activity. Upon S2-S3 or S1-S2 transition, changes occur in the ligand environment of the Mn cluster of WOC influencing the induction of luminol CL.

Orbital Configuration of the Valence Electrons, Ligand Field Symmetry, and Manganese Oxidation States of the Photosynthetic Water Oxidizing Complex: Analysis of the S(2) State Multiline EPR Signals.

A theoretical framework is presented for analysis of all three "multiline" EPR spectra (MLS) arising from the tetramanganese (Mn(4)) cluster in the S(2) oxidation state of the photosynthetic water oxidizing complex (WOC). Accurate simulations are presented which include anisotropy of the g and (four) (55)Mn hyperfine tensors, chosen according to a database of (55)Mn(III) and (55)Mn(IV) hyperfine tensors obtained previously using unbiased least-squares spectral fitting routines. In view of the large (30%) anisotropy common to Mn(III) hyperfine tensors in all complexes, previous MLS simulations which have assumed isotropic hyperfine constants have required physically unrealistic parameters. A simple model is found which offers good simulations of both the native "19-21-line" MLS and the "26-line" NH(3)-bound form of the MLS. Both a dimer-of-dimers and distorted-trigonal magnetic models are examined to describe the symmetry of the Heisenberg exchange interactions within the Mn(4) cluster and thus define the initial electronic basis states of the cluster. The effect of rhombic symmetry distortions is explicitly considered. Both magnetic models correspond to one of several possible structural models for the Mn(4) cluster proposed independently from Mn EXAFS studies. Simulated MLS were constructed for each of the eight (or seven) doublet states of the Mn(4) cluster in the WOC for the two viable oxidation models (3Mn(III)-1Mn(IV) or 3Mn(IV)-1Mn(III)), and using a wide range of axial Mn hyperfine tensors, with either coaxial or orthogonal tensor alignments. We find accurate simulations using the 3Mn(III)-1Mn(IV) oxidation model. In the dimer-of-dimers coupling model, the spin state conversion between two doublet states |S(12),S(34),S(T)|(7)/(2),4,(1)/(2)> and |(7)/(2),3,(1)/(2)> is found to explain the large (25%) contraction in the hyperfine splitting observed upon conversion from the native MLS to the NH(3)-bound MLS. Stabilization of this excited state as the new ground state is caused by change in the intermanganese exchange coupling, without appreciable change in the intrinsic hyperfine tensors. The lack of good simulations of the Ca(2+)-depleted MLS suggests that Ca(2+)-depletion changes both Mn ligation and intermanganese exchange coupling. The 3Mn(IV)-1Mn(III) oxidation model is disfavored because only approximate simulations could be found for the native MLS and no agreement with the NH(3)-bound MLS was obtained. The scalar part of the hyperfine tensors for both Mn(III) and Mn(IV) ions were found to approximate (+/-5%) the values for the dimanganese(III,IV) catalase enzyme, suggesting similar overall ligand types. However, the large (30%) anisotropic part of the Mn(III) hyperfine interaction is opposite in sign to that found in all tetragonally extended six-coordinate Mn(III) ions (i.e., the usual Jahn-Teller splitting). The distribution of spin density from the high-spin d(4) electron configuration of each Mn(III) ion corresponds to a flattened (oblate) ellipsoid. This electronic distribution is favored in five-coordinate ligand fields having trigonally compressed bipyramidal geometry, but it could also arise, in principle, in strained six-coordinate ligand fields having tetragonally compressed geometry, i.e. [Mn(2)(&mgr;-O)](4+) (reverse Jahn-Teller distortion). The resulting valence electronic configurations are described as e'(2)e"(2) and (d(pi))(3)(d(x)()()2(-)(y)()()2)(1), respectively, in contrast to the (d(pi))(3)(d(z)()()2)(1) configuration common to unstrained six-coordinate tetragonally-extended Mn(III) ions, such as found in the [Mn(2)(&mgr;-O)(2)](3+) core in several synthetic dimers and catalase. Both of the former geometries predict strongly oxidizing Mn(III) ions, thereby suggesting a structural basis for the oxidative reactivity of the Mn(4) cluster in the WOC. The magnetic model needed to explain the MLS is not readily reconciled with the simplest structural and electronic models deduced from EXAFS studies of the WOC.

High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation: proton equilibrium, cations, and electrostatics.

The kinetics of pulsed-light photoactivation, the light-induced reassembly of the water-oxidizing complex (WOC) of PSII in the presence of essential inorganic cofactors, has been studied using two improvements: a new efficient chelator, N,N,N',N'-tetrapropionato-1,3-bis(aminomethyl)benzene (TPDBA), for complete extraction of {Mn4} and Ca2+ and an ultrasensitive polarographic cell for O2 detection [Ananyev, G.M., & Dismukes, G.C. (1996) Biochemistry 35, 4102-4109]. Measurements have been made of the initial half-time, t1/2 (sum of the lag time for formation of the first intermediate, IM1, plus the half-time for formation of the second intermediate, IM2), and the steady-state yield, Yss, for recovery of O2 evolution (proportional to the number of active centers). The following conclusions have been reached: (1) cations (Ca2+, Mg2+, and Na+) slow the rate of photoactivation, even though Ca2+ is essential for activity. Two distinct mechanisms appear to be involved: binding to one or both of the first two Mn(2+)-specific sites and screening of negative charges on apo-WOC that are responsible for concentrating Mn2+ ions by electrostatic steering; (2) the Michaelis constant for the calcium requirement for Yss at sufficiently low Mn2+ concentrations (8 microM) that competition at the calcium site does not occur is K(m) = 1.4 mM. Numerically, K(m) is the same for reactivation of O2 evolution in Ca-depleted PSII membranes which retain four Mn ions; (3) in the absence of Ca2+ but in the presence of saturating amounts of Mn2+ (8 Mn/apo-WOC) and Cl-(35 mM) assembly of a stable tetra-Mn cluster occurs neither under illumination nor in the dark after subsequent addition of CaCl2. However, in the presence of suboptimal concentrations of calcium required for maximum Yss, calcium-dependent assembly of stable yet inactive clusters occurs in the light; (4) protons in equilibrium with the buffer greatly increase the half-time 3-fold between pH 6.75 and 5.4, indicating ionization of one or more protons from the first photo-oxidized intermediate formed prior to the rate-limiting step (photo-oxidation of the second Mn2+); (5) the lipophilic membrane soluble anion tetraphenylboron (TPB-), a known reductant of intact WOC, increases the half-time 2.5-fold (< or = 40 microM) and paradoxically stimulates Yss by 50% at 20 microM concentration. These results suggest that TPB- increases the local concentration of Mn2+ adjacent to apo-WOC (Yss increase), while also reducing the S2 and S3 states of the intact WOC at higher concentrations (t1/2 increase). The effects of anions and cations indicates that overcoming the surface potential of the membrane/protein PSII complex may play an important role in the kinetics of reassembly of the {Mn4} cluster; (6) the ratio Y4/Y3 in the kinetics of O2 evolution from a series of single-turnover flashes, a ratio that typically reflects the probability of misses (alpha), grows noticeably larger with increasing extent of recovery of O2 evolving activity and also with increase in the amount of Mn2+, indicating competition between substrate water and excess Mn2+ for reduction of the functional {Mn4} cluster. On the basis of these results, we extend the model for photoactivation to include the antagonistic effects of H+ and Ca2+ in the formation of the first two intermediates.

Electronic and steric effects in manganese Schiff-base complexes as models for the water oxidation complex in photosystem II. The isolation of manganese-(II) and -(III) complexes of 3- and 3,5-substituted N,N′-bis(salicylidene)ethane-1,2-diamine (H2salen) ligands

Manganese-(II) and -(III) complexes of substituted N,N′-bis(salicylidene)ethane-1,2-diamine (H2salen) ligands H2L (substituents are in the 3, 5 or 3,5 positions of the phenyl rings of the salen moiety) have been prepared and thoroughly characterised. The reaction of Mn(ClO4)2·6H2O with H2L in ethanol in air normally leads to manganese(III) complexes ligated by both the N2O2 ligand and water molecule(s). However, by employing electron-withdrawing substituents on the ligand, e.g. 3-Br,5-NO2, a manganese(II) complex can be obtained. A ‘borderline’ ligand is represented by the 5-NO2 derivative (nsalen), which produces a manganese(II) complex contaminated with a small amount of a manganese(III) species. Using a more rigorous oxidising agent in the synthesis, [Fe(η-C5H5)2][FeCl4], drives the reaction totally to a manganese(III) complex [Mn(nsalen)Cl(H2O)]. In addition to magnetic susceptibility studies, cyclic voltammetry has been employed. All the complexes exhibit an oxidation and reduction peak, the reversible character being confirmed by pulse voltammetry. Pulse voltammetry also confirmed the nature of the manganese(II) species [Mn(bnsalen)(H2O)2·2H2O [H2bnsalen =N,N′-bis(3-bromo-5-nitrosalicylidene)ethane-1,2-diamine] and that a slight amount of a manganese(III) species is present in [Mn(nsalen)(H2O)2]·2H2O. Six complexes have been crystallographically characterised. Despite the retention of an octahedral manganese environment in all of them, the supramolecular structures exhibit a wide diversity. The 3,5-dichloro and 5-bromo salen complexes containing co-ordinated water display combined π and hydrogen bonding, as well as dimerisation. The complex [{Mn(µ-dbsalen)(µ-O)}2](dbsalen = 3,5-dibromo derivative) offers an alternative bridging arrangement, and [Mn(bsalen)(MeOH)(OClO3)]·H2O (bsalen = 5-bromo derivative) highlights the versatility of the manganese centre in these systems where, unexpectedly, perchlorate is co-ordinated in place of a lattice water. A more subtle rearrangement of supramolecular structure is obtained in [Mn(nsalen)Cl(H2O)] where the usual combination of π- or hydrogen-bonding interaction is modified by the corresponding ability of the 5-NO2 substituent.