The importance of protein-protein interactions for optimising oxygen activity in photosystem II: reconstitution with a recombinant thioredoxin--manganese stabilising protein.

In this paper we describe how photosystem II (PSII) from higher plants, which have been depleted, of the extrinsic proteins can be reconstituted with a chimeric fusion protein comprising thioredoxin from Escherichia coli and the manganese stabilising protein from Thermosynechococcus elongatus. Surprisingly, even though E. coli thioredoxin is completely unrelated to PSII, the fusion protein restores higher rates of activity upon rebinding to PSII than either the native spinach MSP, or T. elongatus MSP. PSII reconstituted with the fusion protein also has a lower requirement for calcium than PSII with the small extrinsic proteins removed, or PSII reconstituted with spinach or T. elongatus MSP. The MSP portion of the fusion protein is less thermally stable compared to isolated MSP from T. elongatus, which could be the key to its superior activation capability through greater flexibility. This work reveals the importance of protein-protein interactions in the water splitting activity of PSII and suggests that conformational configurations, which increase flexibility in MSP, are essential to its function, even when these are induced by an unrelated protein.

A quantitative assessment of the carbonic anhydrase activity in photosystem II.

Using a carbonic anhydrase assay based on membrane inlet mass spectrometry (MIMS), we have extended our earlier investigations of Photosystem II (PSII)-associated carbonic anhydrase activity in spinach PSII preparations (W. Hillier, I. McConnell, M. R. Badger, A. Boussac, V.V. Klimov G. C. Dismukes, T. Wydrzynski Biochemistry 2006, 45:2094). The relationship between the carbonic anhydrase activity and O(2) evolution has been evaluated in terms of the effects of metal ion addition, preparation type, light, and response to specific inhibitors. The results indicate that the PSII-associated carbonic anhydrase activity is variable and appears not to be associated specifically with the oxygen evolving activity nor the 33 kDa extrinsic manganese stabilising protein.

Substrate water exchange in photosystem II depends on the peripheral proteins.

The (18)O exchange rates for the substrate water bound in the S(3) state were determined in different photosystem II sample types using time-resolved mass spectrometry. The samples included thylakoid membranes, salt-washed Triton X-100-prepared membrane fragments, and purified core complexes from spinach and cyanobacteria. For each sample type, two kinetically distinct isotopic exchange rates could be resolved, indicating that the biphasic exchange behavior for the substrate water is inherent to the O(2)-evolving catalytic site in the S(3) state. However, the fast phase of exchange became somewhat slower (by a factor of approximately 2) in NaCl-washed membrane fragments and core complexes from spinach in which the 16- and 23-kDa extrinsic proteins have been removed, compared with the corresponding rate for the intact samples. For CaCl(2)-washed membrane fragments in which the 33-kDa manganese stabilizing protein (MSP) has also been removed, the fast phase of exchange slowed down even further (by a factor of approximately 3). Interestingly, the slow phase of exchange was little affected in the samples from spinach. For core complexes prepared from Synechocystis PCC 6803 and Synechococcus elongatus, the fast and slow exchange rates were variously affected. Nevertheless, within the experimental error, nearly the same exchange rates were measured for thylakoid samples made from wild type and an MSP-lacking mutant of Synechocystis PCC 6803. This result could indicate that the MSP has a slightly different function in eukaryotic organisms compared with prokaryotic organisms. In all samples, however, the differences in the exchange rates are relatively small. Such small differences are unlikely to arise from major changes in the metal-ligand structure at the catalytic site. Rather, the observed differences may reflect subtle long range effects in which the exchange reaction coordinates become slightly altered. We discuss the results in terms of solvent penetration into photosystem II and the regional dielectric around the catalytic site.

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.

The affinities for the two substrate water binding sites in the O(2) evolving complex of photosystem II vary independently during S-state turnover.

The first determinations of substrate water binding to the O(2) evolving complex in photosystem II as a complete function of the S states have been made. H(2)(18)O was rapidly injected into spinach thylakoid samples preset in either the S(0), S(1), S(2), or S(3) states, and the rate of (18)O incorporation into the O(2) produced was determined by time-resolved mass spectrometry. For measurements at m/e = 34 (i.e., for the (16)O(18)O product), the rate of (18)O incorporation in all S states shows biphasic kinetics, reflecting the binding of the two substrate water molecules to the catalytic site. The slow phase kinetics yield rate constants at 10 degrees C of 8 +/- 2, 0.021 +/- 0.002, 2.2 +/- 0.3, and 1.9 +/- 0.2 s(-1) for the S(0), S(1), S(2), and S(3) states, respectively, while the fast phase kinetics yield a rate constant of 36.8 +/- 1.9 s(-1) for the S(3) state but remain unresolvable (>100 (s-1)) for the S(0), S(1), and S(2) states. Comparisons of the (18)O exchange rates reveal that the binding affinity for one of the substrate water molecules first increases during the S(0) to S(1) transition, then decreases during the S(1) to S(2) transition, but stays the same during the S(2) to S(3) transition, while the binding affinity for the second substrate water molecule undergoes at least a 5-fold increase on the S(2) to S(3) transition. These findings are discussed in terms of two independent Mn(III) substrate binding sites within the O(2) evolving complex which are separate from the component that accumulates the oxidizing equivalents. One of the Mn(III) sites may only first bind a substrate water molecule during the S(2) to S(3) transition.

Kinetic determination of the fast exchanging substrate water molecule in the S3 state of photosystem II.

In a previous communication we showed from rapid isotopic exchange measurements that the exchangeability of the substrate water at the water oxidation catalytic site in the S3 state undergoes biphasic kinetics although the fast phase could not be fully resolved at that time [Messinger, J., Badger, M., and Wydrzynski, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3209-3213]. We have since improved the time resolution for these measurements by a further factor of 3 and report here the first detailed kinetics for the fast phase of exchange. First-order exchange kinetics were determined from mass spectrometric measurements of photogenerated O2 as a function of time after injection of H218O into spinach thylakoid samples preset in the S3 state at 10 degreesC. For measurements made at m/e = 34 (i. e., for the mixed labeled 16,18O2 product), the two kinetic components are observed: a slow component with k1 = 2.2 +/- 0.1 s-1 (t1/2 approximately 315 ms) and a fast component with k2 = 38 +/- 4 s-1 (t1/2 approximately 18 ms). When the isotopic exchange is measured at m/e = 36 (i.e., for the double labeled 18,18O2 product), only the slow component (k1) is observed, clearly indicating that the substrate water undergoing slow isotopic exchange provides the rate-limiting step in the formation of the double labeled 18,18O2 product. When the isotopic exchange is measured as a function of temperature, the two kinetic components reveal different temperature dependencies in which k1 increases by a factor of 10 over the range 0-20 degreesC while k2 increases by only a factor of 3. Assuming simple Arrhenius behavior, the activation energies are estimated to be 78 +/- 10 kJ mol-1 for the slow component and 39 +/- 5 kJ mol-1 for the fast component. The different kinetic components in the 18O isotopic exchange provide firm evidence that the two substrate water molecules undergo separate exchange processes at two different chemical sites in the S3 state, prior to the O2 release step (t1/2 approximately 1 ms at 20 degreesC). The results are discussed in terms of how the substrate water may be bound at two separate metal sites.

Room-temperature vibrational difference spectrum for S2QB-/S1QB of photosystem II determined by time-resolved Fourier transform infrared spectroscopy.

Time-resolved FTIR spectroscopy has been used to kinetically characterize the vibrational properties of intact photosystem II-enriched membrane samples undergoing the S1QB-to-S2QB- transition at room temperature. To optimize the experimental conditions for the FTIR measurements, oxygen polarographic and variable chlorophyll a fluorescence measurements were used to define the decay of S2 and QA-, respectively. The flash-induced S2QB-/S1QB difference spectra were measured at a temporal resolution of 4.44 s and a spectral resolution of 4 cm-1. An intense positive band is observed at 1480 cm-1 in the difference spectrum and shows a slow decay with a half time of approximately 13 s. Based on its decay kinetics and analogy to the infrared absorption of QA- of photosystem II and QB- in bacterial reaction centers, we conclude that the 1480 cm-1 band arises from QB- of PSII and tentatively assign it to the upsilon(CO) mode of the semiquinone anion QB-. The infrared spectral features attributed to the S1-to-S2 transition of the Mn cluster at room temperature show striking similarity to the S2/S1 difference spectrum measured at cryogenic temperatures (Noguchi, T., Ono, T.-A., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200).

Role of an extrinsic 33 kilodalton protein of photosystem II in the turnover of the reaction center-binding protein D1 during photoinhibition.

The reaction center-binding protein D1 of photosystem II (PS II) undergoes rapid turnover under light stress conditions. In the present study, we investigated the role of the extrinsic 33 kDa protein (OEC33) in the early stages of D1 turnover. D1 degradation was measured after strong illumination (1000-5000 microE m-2 S-1) of spinach manganese-depleted, PSII-enriched membrane and core samples in the presence and absence of the OEC33 under aerobic conditions at room temperature. PSII samples lacking the OEC33 were prepared by standard biochemical treatments with Tris or CaCl2/NH2OH while samples retaining the OEC33 were prepared with NH2OH or NaCl/NH2OH. The degradation of D1, monitored by SDS/urea-polyacrylamide gel electrophoresis and Western blotting using specific antibodies against D1, proceeds to a greater extent in NH2OH-treated samples than in Tris-treated samples over a 60 min illumination period. Under the same conditions, significantly more aggregation of D1 occurs in the Tris-treated samples than in the NH2OH-treated samples. The lower level of D1 degradation in Tris-treated samples is not due to secondary proteolysis, as judged from the time course for degradation at 25 degrees C or the degradation pattern at 4 degrees C. Similarly, for NaCl/NH2OH-treated samples, D1 degradation is greater and D1 aggregation less than in CaCl2/NH2OH-treated samples. The effect of the presence of the OEC33 on D1 degradation and aggregation is confirmed by reconstitution experiments in which the isolated OEC33 is restored back to Tris-treated samples. During very strong illumination, significant loss of CP43 also occurs in Tris-treated but not in NH2OH-treated samples. Structural analysis of PS II core complexes by Fourier transform infrared (FT-IR) spectroscopy revealed very little change in the protein secondary structure after 10 min illumination of NH2OH-treated samples while a large 10% decrease of alpha-helix content occurs in Tris-treated samples. On the basis of these results, we suggest that either (1) the OEC33 stabilizes the structural integrity of PS II such that it prevents the photodamaged D1 protein from aggregating with nearby polypeptides and thereby facilitating degradation or (2) the OEC33 specifically stabilizes CP43, a putative D1-specific protease, which normally promotes the efficient degradation of D1.

Yz. reduction kinetics in the absence of the manganese-stabilizing protein of photosystem II.

Decay of Signal IIvf of photosystem II (PSII), under repetitive flash conditions, was examined in whole cells of wild-type Synechocystis sp. PCC6803 and in cells of an engineered strain, delta psbO, which lacks the extrinsic 33 kDa manganese-stabilizing protein (MSP). Previous polarographic analysis had shown that O2 release during the S3-->[S4]-->S0 transition of the catalytic cycle is significantly retarded in the delta psbO strain relative to the wild-type [Burnap et al. (1992) Biochemistry 31, 7404-7410]. The present experiments provide evidence that a parallel retardation in the rate of reduction of photooxidized Yz by the H2O oxidation complex is due to the absence of MSP. The half-time of the Signal IIvf component, corresponding to Yz. reduction during the S3-->[S4]-->S0 transition, was estimated to be 1.2 and 6.0 ms in the wild-type and delta psbO cells, respectively.

A time-resolved FTIR difference study of the plastoquinone QA and redox-active tyrosine YZ interactions in photosystem II.

In this paper, we present the first time-dependent measurements of flash-induced infrared difference spectra of photosystem II (PSII) using Fourier transform infrared (FTIR) spectroscopy. With this experimental approach, we were able to obtain the YZoxQA-/YZQA vibrational difference spectrum of Tris-washed, PSII-enriched samples in the absence of hydroxylamine at room temperature (16 +/- 2 degrees C), with a spectral resolution of 4 cm-1 and a temporal resolution of 50 ms. In order to determine the dominant species in the FTIR spectrum at a particular point in time after an excitation flash, the decay kinetics of YZox and QA- were independently monitored by EPR and chlorophyll a fluorescence, respectively, under the same experimental conditions. These measurements confirmed that the addition of DCMU to Tris-washed PSII samples does not significantly affect the YZox decay, but does substantially slow down the QA- decay. By making use of the difference in the decay kinetics using DCMU, the QA-/QA signals could be separated from the YZox/YZ signals and a pure QA-/QA difference spectrum obtained. By comparison of the YZoxQA-/YZQA difference spectrum with the pure QA-/QA difference spectrum, a large differential band at 1706/1699 cm-1 could be identified and associated with YZ oxidation. In contrast, an intense band at 1478 cm-1, whose DCMU-sensitive decay follows the QA- decay based on the chlorophyll a fluorescence measurements, was present in all of the time-resolved spectra. Since no significant reversible Chl+ radicals could be detected by the EPR measurements under our experimental conditions, we confirm that this band most likely arises only from the semiquinone anion QA- [Berthomieu, C., Nabedryk, E., Mäntele, W., & Breton, J. (1990) FEBS Lett. 269, 363-367].

S(-3) state of the water oxidase in photosystem II.

The effect of the reductant hydrazine on the flash-induced oxygen oscillation patterns of spinach thylakoids was used to characterize a new super-reduced redox state of the water oxidase in photosystem II. The formation of a discrete S(-3) state is evident from the shift of the first maximum of oxygen evolution from the 3rd flash through the 5th flash to the 7th flash during a 90 min incubation of dark-adapted thylakoids with 10 mM hydrazine sulfate at pH 6.8 on ice. A distinct period four oscillation with further maxima on the 11th and 15th flashes is still observed at this stage of the incubation. The data analysis within the framework of an extended Kok model reveals that a S(-3) state population of almost 50% can be achieved by this treatment. A prolonged incubation of the S(-3) sample with 10 mM hydrazine (and even 100 mM) does not lead to a further shift of the first maximum toward the 9th flash that could reflect the formation of the S(-5) state. Instead, a slow oxidation of S(-3) to S(-2) takes place by an as yet unidentified electron acceptor. A consistent simulation of all the measured oxygen oscillation patterns of this study could, however, only be achieved by including the formal redox states S(-4) and S(-5) in the fits (S(-4) + S(-5) up to 35%). The implications of these findings for the oxidation states of the manganese in the tetranuclear cluster of the water oxidase are discussed.

Photochemical reactions of photosystem II in ethylene glycol.

The behavior of photosystem II (PSII) reactions was investigated under conditions of decreasing water content by the addition of increasing concentrations of ethylene glycol (EG). The photosynthetic activities were measured for PSII samples either directly in aqueous solutions of EG or in the standard buffer medium following EG treatment. Several effects on PSII arise upon exposure to EG. Below 50% EG there are no significant irreversible changes, although there is a slowing of the QA-reoxidation kinetics in the presence of EG. At concentrations of 50-70% EG, protein structural changes occur that include the release of the 16, 23, and 33 kDa extrinsic proteins and two of the catalytic Mn ions. For these samples, the capacity for O2 evolution is considerably reduced and the formation of donor side H2O2 is enhanced. In 60% EG, the nanosecond components in the rate of P680+ reduction are converted entirely to microsecond kinetics which upon return of the sample to the standard buffer medium are partially restored, indicating that EG has a reversible, solvent effect on the PSII donor side. At concentrations of EG > 70% chlorophyll fluorescence measurements reveal reversible increases in the FO level concomitant with the generation and disappearance of a 5 microseconds decay component in the P680+ reduction kinetics. This result may indicate a solvent-induced uncoupling of the light harvesting pigment bed from the reaction center complex. As the EG concentration is increased to 80-100%, there is an irreversible loss of the primary charge separation. The use of EG as a cryoprotectant and as a water-miscible organic solvent for PSII is discussed.

Detection of one slowly exchanging substrate water molecule in the S3 state of photosystem II.

The exchangeability of the substrate water molecules at the catalytic site of water oxidation in photosystem II has been probed by isotope-exchange measurements using mass spectrometric detection of flash-induced oxygen evolution. A stirred sample chamber was constructed to reduce the lag time between injection of H2(18)O and the detecting flash by a factor of more than 1000 compared to the original experiments by R. Radmer and O. Ollinger [(1986) FEBS Lett. 195, 285-289]. Our data show that there is a slow (t1/2 approximately 500 ms, 10 degrees C) and a fast (t1/2 <25 ms, 10 degrees C) exchanging substrate water molecule in the S3 state of photosystem II. The slow exchange is coupled with an activation energy of about 75 kJ/mol and is discussed in terms of a terminal manganese oxo ligand, while the faster exchanging substrate molecule may represent a water molecule not directly bound to the manganese center.

Photoproduction of hydrogen peroxide in Photosystem II membrane fragments: A comparison of four signals.

The present study describes the formation of different forms of peroxide in Photosystem II (PS II) by using a chemiluminescence detection technique. Four chemiluminescence signals (A, B, C and D) of the luminolperoxidase (Lu-Per) system, which detects peroxide, are found in illuminated O2-evolving Photosystem II (PS II) membrane fragments isolated from spinach. Signal A ('free peroxide') peaking around 0.2-0.3 s after mixing PS II membrane fragments with Lu-Per is eliminated by catalase or removal of oxygen from the suspension and ascribed to O2 interaction with reduced PS II electron acceptors. In contrast, signal B peaking around 1.5 min remains largely unaffected under anaerobic conditions, as well as in the presence of catalase (20 µg/ml). Under flash illumination the extent of this signal exhibits a weak period four oscillation (maximum at third and 7th flash). Its yield increases up to the third flash, but is close to zero in the fourth flash. An analogous behaviour is observed in flashes 5 to 8. Signal B is ascribed to Lu-Per interaction with the water-oxidizing system being in S2 and/or S3-state. Signal C ('bound peroxide') detected as free peroxide after acid decomposition of illuminated PS II particles is observed on the 1 st flash and oscillates with period 2 with superposition of period 4. It is evidently related to peroxide either released from S2 or formed at S2 upon acid shock treatment. Signal D ('slowly released peroxide') peaking around 2-3 s after mixing is observed in samples after various treatments (LCC-incubation, washing with 1 M NaCl at pH 8 or with 1 M CaCl2, Cl(-)-depletion) that lead to at least partial removal of the extrinsic proteins of 18, 24 and 33 kDa without Mn extraction. The average amplitude of this signal corresponds with a yield of about 0.2 H2O2 molecules per RC and flash. In a flash train, the extent of signal D exhibits an oscillation pattern with a minimum at the 3rd flash. We assume that these treatments increase the release of 'bound' peroxide (upon injection into the Lu-Per assay) either formed in the normal oxidative pathway of the water oxidase in the S2 or the S3-state or give rise to peroxide formation due to higher accessibility of the Mn-cluster to water molecules.

Increases in peroxide formation by the Photosystem II oxygen evolving reactions upon removal of the extrinsic 16, 22 and 33 kDa proteins are reversed by CaCl2 addition.

This communication introduces a new spectrophotometric assay for the detection of peroxide generated by Photosystem II (PS II) under steady state illumination in the presence of an electron acceptor. The assay is based on the formation of an indamine dye in a horseradish peroxidase coupled reaction between 3-(dimethylamino)benzoic acid and 3-methyl-2-benzothiazolinone hydrazone. Using this assay, we found that as the O2 evolution activity of PS II-enriched membrane fragments is decreased by treatments which cause the dissociation of the 33 and/or 23 and 16 kDa extrinsic proteins (i.e., CaCl2-washing, NaCl-washing, lauroylcholine-treatment and ethylene glycol-treatment), light-induced peroxide formation increases. Both the losses of O2 evolution and increases in peroxide formation seen under these conditions are reversed by CaCl2 addition, indicating that the two activities originate from the water-splitting site. However, the increased rates of peroxide formation do not quantitatively match the losses in O2 evolution activity. We suggest that a rapid consumption of the peroxide takes place via a catalase/peroxidase activity at the water-splitting site which competes with both the O2 evolution and peroxide formation reactions. The observed peroxide formation is interpreted as arising from enhanced water accessibility to the catalytic site upon perturbation of the extrinsic proteins which then leads to alternate water oxidation side reactions.

Anti-sense RNA efficiently inhibits formation of the 10 kd polypeptide of photosystem II in transgenic potato plants: analysis of the role of the 10 kd protein.

A chimeric gene encoding an anti-sense RNA of the 10 kd protein of the water-splitting apparatus of photosystem II of higher plants under the control of the CaMV 35S promoter was introduced into potato using Agrobacterium based vectors. The expression of the anti-sense RNA led to a significant reduction of the amounts of the 10 kd protein and RNA in a number of transgenic plants. In three out of 36 plants tested, the level of the 10 kd protein was only up to 1-3% compared with the wild-type control. The drastic reduction of the 10 kd protein did not influence the accumulation of other photosystem II associated polypeptides at both the RNA and protein level. Furthermore no phenotypic differences were observed between potato plants expressing wild-type and drastically reduced levels of the 10 kd protein with respect to growth rate, habitus or ultrastructure of the chloroplasts. Measurements of the relaxation of the flash-induced enhancement in the fluorescence quantum yield as determined in intact leaves and the rates and characteristic oscillation pattern of O2 evolution as determined in isolated thylakoid samples however, show that the elimination of the 10 kd protein on the one hand retards reoxidation of QA- and on the other hand introduces a general disorder into the PSII complex.

Is there a direct chloride cofactor requirement in the oxygen-evolving reactions of photosystem II?

The dark incubation at room temperature of photosystem II (PS II) membrane fragments in a chloride-free medium at pH 6.3 slowly leads to large chloride-restorable and non-restorable O2 evolution activity losses with time as compared with control samples incubated in the presence of 10 mM NaCl. The chloride requirement in O2 evolution generated under these conditions reveals a complex interplay among various experimental parameters, including the source of the plant material, the times of incubation, the sample concentration, the chloride concentration, as well as those treatments which are believed to specifically displace chloride from PS II such as alkaline pH pretreatment and Na2SO4 addition. The results indicate that secondary, structural changes within the PS II complex are an important factor in determining the influence of chloride on the O2 evolution activity and raise the question whether or not chloride ions actually play a direct cofactor role in the water-oxidizing reactions leading to O2 evolution.

Current perceptions of Photosystem II.

In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385-398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q400 component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.

Periodic changes in the oxidation state of manganese in photosynthetic oxygen evolution upon illumination with flashes.

The pattern of manganese released from chloroplast membranes by a rapid temperature shock after various illumination regimes indicates that changes in the oxidation state of bound manganese occur during photosynthesis. Continuous illumination decreases by 35-40% the amount of Mn(II) released in the presence of K3Fe(CN)6 compared with a dark-adapted control. Following illumination and heat treatment, the addition of the reductant H2O2 to the samples causes an increase in the level of electron paramagnetic resonance (EPR)-detectable manganese. The pH dependence of the H2O2 reduction indicates that the non-EPR-detectable manganese present in the heated sample after illumination is in the form of higher oxidation state compounds, e.g. MnO2. The light-induced Mn(II) decrease is reversible in the dark with t 1/2 approx. 40 s and can be prevented by the presence of the Photosystem II inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethyl urea or fluorocarbonylcyanide phenylhydrazone during the illumination period. After a series of brief flashes of light the Mn(II) released by heat treatment oscillates over periods of four flashes. The pattern is similar to the O2 yield flash pattern and suggests that a cycling of manganese oxidation states is involved in the O2 evolution mechanism. The oscillations in the Mn(II) release are analyzed in terms of the current four-step model for O2 evolution. The analysis suggests that manganese is successively oxidized in the first two steps, but undergoes a partial reduction on the third step. This result is consistent with the concept that water undergoes a partial oxidation prior to the release of O2 from the water-splitting complex.

Nuclear magnetic relaxation by the manganese in aqueous suspensions of chloroplasts.

Proton and oxygen-17 NMR relaxation rate (T1-1 and T2-1) data are presented for aqueous suspensions of dark-adapted chloroplasts. It is concluded from the dependence of the proton relaxation rates (PRR) upon Mn concentration that T1-1 and T2-1 are determined largely by the loosely bound Mn present in the chloroplast membranes. The frequency and temperature dependences of PRR are characteristic of Mn(II). The effects of oxidants (e.g., ferricyanide) and reductants (e.g., tetraphenylboron) on the PRR indicate that only about one-third to one-fourth of the loosely bound Mn is present in the dark-adapted chloroplasts as Mn(II), the remainder being in a higher oxidation state(s), probably Mn(III). The frequency dependence of the PRR for the chloroplast suspensions was fitted by a simplified form of the Solomon-Bloembergen-Morgan equations, and the following parameters were obtained: tauS = (1.1 +/- 0.1) X 10(-8) S; tauM = (2.2 +/- 0.2) X 10(-8) S; and B = (0.9 +/- 0.09) X 10(19). The oxygen-17 T1 and T2 data for suspensions before and after treatment with a detergent are consistent with the location of the manganese in the interior of the thylakoids. An analysis of the relaxation rates shows that the average lifetime of a water molecule inside a thylakoid is greater than 1 ms.