Binding of the inhibitor NH3 to the oxygen-evolving apparatus of spinach chloroplasts.

Experiments are described on flash-induced luminescence of isolated spinach chloroplasts after addition of NH4Cl. The results indicate a binding of NH3, presumably in competition with water, in the oxidation states S2 and S3, i.e. the states reached upon illumination of dark-adapted material with one and two flashes, respectively. In the initial state S1, no binding of NH3 occurs. In state S2 the binding of ammonia is rapid (half-time about 0.5 s) and rapidly reversible; in state S3 the binding is slower (half-time about 10 s) and slowly reversible. NH3 bound to S4 prevents the oxidation of water. NH3 bound to S2 decreases the rate of the back reaction of reduced primary acceptor (Q-), indicating a charge stabilization, i.e. a decrease in the redox potential of S2 due to interaction with ammonia. In Tris-washed chloroplasts, the stability of the positive charge generated in a flash is much smaller than in normal chloroplasts and not increased by NH3. On the basis of these observations it is postulated that, in the absence of NH3, states S2 and S3 are stabilized by manganese-coordinated, bound water.     

The interaction of ammonia with the photosynthetic oxygen-evolving system

The reaction of ammonia with the oxygen-evolving system was investigated using EPR. Two sites with distinct binding properties were found. One site, previously known to be responsible for the modification by ammonia of the multiline EPR signal from the S₂ state and believed to be accessible in this state only, was found to bind ammonia also in the S₁ state although weaker. The second binding site, identified by the effect of bound ammonia on the shape and position of the g = 4.1 EPR signal, was also found to be accessible in both the S₁ and S₂ states. The apparent dissociation constants for ammonia at the two sites in the S₁ and S₂ states were determined. In neither state did the binding the ammonia account for the observed inhibition of oxygen evolution, suggesting that binding to other S states plays an important role in the inhibition. Chloride, which is known to interfere with ammonia-induced inhibition of oxygen evolution, was found to compete with ammonia at the site associated with the modification of the g = 4.1 EPR signal. The broadening of the hyperfine lines of the multiline EPR signal, seen in the presence of ¹⁷O-labeled water, was still observed after the modification of the signal by ammonia. This indicates that ammonia has not completely displaced water bound to the catalytic site in the S₂ state. The results of the binding studies are interpreted in terms of a two state — two site model, where the two states are identified by their EPR signals, the multiline and the g = 4.1 signal, respectively, and the two sites identified by the effects of ammonia on these signals and where the equilibrium between the two states is regulated by the binding of ligands to the sites.     

Effect of low temperature (−30 to −60°C) on the reoxidation of the Photosystem II primary electron acceptor in the presence and absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea

The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q⁻ to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center:

1. (1) In dark-adapted chloroplasts (i.e. in States S₀+S₁ according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.

2. (2) When chloroplasts are placed in the S₂+S₃ states by a two-flash preillumination at room temperature, the reoxidation of Q⁻ after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.

3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O₂ molecule.

4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.

The S-state dependence of Cl binding to plant Photosystem II

A combined single-turnover flash and ³⁵Cl NMR technique has been used to monitor S-state dependence of Cl⁻ binding to PS-II particles derived from mangrove (Avicennia marina). No detectable high-affinity binding was found to particles in the S₀ and S₁ states, but binding with an affinity comparable to that which activates O₂ evolution was found in the S₂ and S₃ states.     

Studies on the mechanism of destabilization of the positive charges trapped in the photosynthetic water-splitting enzyme system Y by a deactivation-accelerating agent

The mechanism of the 2-(3,4,5-trichloro)anilino-3,5 dinitrothiophene (ANT 2S)-induced cyclic electron flow leading to the discharge of the higher-trapped-hole accumulation states S₂ and S₃ in the photosynthetic water-splitting enzyme system Y of chloroplasts has been investigated. It was found:

1. 1. Under normal conditions the ANT 2s-catalyzed cycle includes both light reactions.

2. 2. By selective kinetical inhibition of the electron flow through P700—either by histone treatment or by 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone blockage—the ANT 2s-induced deactivation of S₂ and S₃ is not significantly changed. Hence, System I activity is not a functional prerequisite for the ANT 2s-catalyzed discharge of S₂ and S₃.

3. 3. The reciprocal half time of the ANT 2s-induced decay of the relative average oxygen yield per flash, as a function of the time t_d between the flashes representing the degree of the Acceleration of the Deactivation Reactions of the water-splitting enzyme system (ADRY) effect, is nearly linearly related to the ANT 2s concentration within the range of 10⁻⁷–10⁻⁶ M.

4. 4. In respect to the mode of action of ANT 2s two different types of mechanism have been discussed: fixed-place mechanism and mobile-catalyst mechanism.

5. 5. Based on the experimental data the conclusion has been drawn that the ADRY agent ANT 2s probably acts as a mobile catalyst.

Studies on the ADRY agent-induced mechanism of the discharge of the holes trapped in the photosynthetic waterspliting enzyme system Y

The effect of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p) on the oxygen evolution, fluorescence and delayed light emission of spinach chloroplasts has been investigated. It was found that;

1. 1. ANT 2p strongly accelerates the deactivation of states S₂ and S₃ of the water-splitting enzyme system Y.

2. 2. In DCMU-poisoned chloroplasts ANT 2p prevents the back reaction of the electrons located at the primary acceptor, Q, with the holes (positive charges) stored in the water-splitting enzyme system Y.

3. 3. In chloroplast suspensions without artificial electron acceptors, the fluorescence rise in weak actinic light vanishes in the presence of ANT 2p. The fluorescence yield in DCMU-inhibited chloroplasts is not significantly changed by ANT 2p.

4. 4. The intensity of the delayed light emitted after excitation with one short flash is remarkably decreased by ANT 2p.

5. 5. In weak actinic light the reduction rate of the artificial electron acceptor methyl viologen is suppressed in the presence of ANT 2p.

From these experimental results it is concluded that ANT 2p induces a cycle within the electron transport chain, leading to a dissipative recombination of the holes stored in the water-splitting enzyme Y with the electrons of an as yet unknown donor.

Two possibilities for the mode of action of this cycle are discussed.     

Thermoluminescence as a probe of photosystem II. The redox and protonation states of the secondary acceptor quinone and the O2-evolving enzyme

The thermoluminescence band observed in chloroplasts after flash excitation at ambient temperatures has recently been identified as being due to recombination of the electron on the semiquinone form of the secondary plastoquinone acceptor, Q_B, with positive charges on the oxygen-evolving enzyme, S₂ and S₃ (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). Further investigation of this thermoluminescence confirms this assignment and provides information on the function of PS II. The following data are reported: (1) Washing of chloroplasts with ferricyanide lowers the concentration of Q_B⁻ in the dark and predictable changes in the extent of the thermoluminescence band are observed. (2) The thermoluminescence intensity arising from S₂Q_B⁻ is approximately one half of that arising from S₃Q_B⁻. (3) Preflash treatment followed by dark adaptation results in changes in the intensity of the thermoluminescence band recorded after a series of flashes. These changes can be explained according to the above assignments for the origin of the thermoluminescence and if Q_B⁻ provides an important source of deactivating electrons for the S states. Computer simulations of the preflash data are reported using the above assumptions. Previously unexplained data already in the literature (Läufer, A. and Inoue, Y. (1980) Photobiochem. Photobiophys. 1, 339–346) can be satisfactorily explained and are simulated using the above assumptions. (4) Lowering the pH to pH 5.5 results in a shift of the S₂Q_B⁻ thermoluminescence band to higher temperatures while that arising from S₃Q_B⁻ does not shift. This effect is interpreted as indicating that Q_B⁻ is protonated and the S₂ to S₃ reaction involves deprotonation while the S₁ to S₂ reaction does not.     

脱甲河水系CH4关键产生途径及其稳定碳同位素特征

为明确脱甲河溶存CH₄关键产生途径,明晰水系碳同位素组成及其分布特征,为小流域CH₄排放估算和减排提供数据支撑.利用双层扩散模型法估算了CH₄浓度和传输通量,研究了周年内脱甲河4级河段(S₁～S₄)水体CH₄通量的时空分布及其主控环境因子;运用稳定同位素方法探究了溶存CH₄关键产生途径,分析了溶解CH₄、悬浮颗粒物和沉积物有机质δ¹³C分布特征.结果表明: 水体pH均值为(7.27±0.03),各河段四季差异均显著;溶解氧(DO)在0.43～13.99 mg·L^-1内变化,S₁河段DO浓度最高且夏、秋季差异显著,其他河段均为冬与春、夏、秋季差异显著;可溶性有机碳(DOC)变化范围是0.34～8.32 mg·L^-1,由S₁至S₄河段总体呈递增趋势;水体电导率(EC)和氧化还原电位(ORP)变化范围分别是17～436 μS·cm^-1和-52.30～674.10 mV,各河段差异明显;铵态氮(NH₄⁺-N)、硝态氮(NO₃^--N)浓度分别在0.30～1.35(平均0.90±0.10) mg·L^-1和0.82～2.45 (平均1.62±0.16) mg·L^-1内变化.溶存CH₄浓度和传输通量变化范围分别是0～5.28 (平均0.46±0.06) μmol·L^-1和-0.34～619.72 (平均53.88±7.15) μg C·m^-2·h^-1;均存在时空变化且变异规律相似,为春季>冬季>夏季>秋季,S₂>S₃>S₄>S₁.通量与水体铵态氮和DOC浓度均呈显著正相关.各级河段均以乙酸发酵产甲烷途径为主导,但不同河段差异明显,乙酸发酵途径产CH₄贡献率以S₁河段最高(87%),其次为S₄(81%),S₂、S₃分别达到78%和76%.溶存CH₄、悬浮颗粒物和沉积物有机质的δ¹³C均值分别为-41.64‰±1.91‰、-14.07‰±1.06‰和-26.20‰±1.02‰,溶存甲烷δ¹³C与沉积物有机质的δ¹³C呈显著正相关,与其传输通量呈极显著负相关.     

A study of dark luminescence in Chlorella. Background luminescence, 3-(3,4-dichlorophenyl)-1,1-dimethylurea-triggered luminescence and hydrogen peroxide chemiluminescence

Dark luminescence, defined as the ability of completely relaxed (darkadapted) photosynthetic systems to emit light, has been studied in Chlorella. Three main effects have been demonstrated. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea elicits a weak emission L_D of very long lifetime (several minutes); it is believed to result from a negative shift of redox potential of the secondary System II electron acceptor B producing in some centers a state Q⁻ (reduced primary acceptor), as postulated by Velthuys and Amesz ((1974) Biochim. Biophys. Acta 333, 85–94), which can recombine with an oxidizing equivalent in a state S₂ present in very small amount. As in photoinduced luminescence, this recombination excites chlorophyll which then emits light. A much stronger emission L_H is observed after injection of H₂O₂. Both signals are modified or suppressed by treatments specific of the oxygen emission system, such as: thermal denaturation at 50°C, NH₂OH, etc. In addition, a weak, permanent background luminescence L₀ has been observed; like L_D and L_H, it is a System II property and requires the integrity of the oxygen-evolving system. It is believed to reflect a very slow back flow of electrons from an endogeneous reductant pool to oxygen through part of the photosynthetic chain. Using flash preillumination, it is demonstrated that H₂O₂ is able to oxidize S₀ into S₂, the latter giving rise to L_H; H₂O₂ does not act on S₁ (or much less). The reactive site of H₂O₂ seems to be the same as the binding site of NH₂OH. Evidence is given that the strong L_H signal in particular reveals a stable, low pH of the intrathylakoid phase in Chlorella.     

Limiting steps in Photosystem II and water decomposition in Chlorella and spinach chloroplasts

1. 1. By varying the redox potential of a chloroplast suspension, we obtained new evidence for an equilibrium between states S₀ and S₁ in the model of Kok, B., Forbush, B. and McGloin, N. (1970, Photochem. Photobiol. 11, 457–475). The mid-point potential of the S₀ to S₁ couple is close to that for the pool of the electron acceptor of System II, A to A⁻.

2. 2. The limiting steps between two consecutive photoreactions of System II in Chlorella and spinach chloroplasts, have been studied.

2.1. (a) The limiting step from S₁ to S₂ (noted γ₁ (Δt)) is not exponential. Its temperature coefficient becomes greater as the reaction proceeds. The shape of the kinetics is an intrinsic property of each center. Chloroplasts fixed with 2% glutaraldehyde, show simple first order kinetics.

2.2. (b) The limiting step from S₀ to S₁ (γ₀ (Δt)) exhibits the same characteristics as γ₁ (Δt)).

2.3. (c) The limiting step from S₂ to S₃ (γ₂ (Δt)) shows sigmoidal kinetics; two reactions are involved. One of the reactions exhibits the same properties as γ₀ (Δt) and γ₁ (Δt).

2.4. (d) The limiting step from S₃ to S₀ (γ₃ (Δt)) is a first order reaction, two times slower than the other transitions. This reaction is interpretated in terms of oxygen release.

3. 3. We also studied the limiting steps in the presence of low concentrations (50 μM) of hydroxylamine. The results favor the binding of two molecules of hydroxylamine to every photochemical center.

日光温室番茄-西瓜轮作系统不同水氮处理氨挥发特征

为探究黄土高原地区日光温室果蔬栽培中氨挥发特征,在陕西省杨凌区选择当地典型的日光温室,设置4个不同的水氮处理,采用密闭式间歇抽气法监测番茄-西瓜轮作季的氨挥发特征.结果表明: 日光温室栽培土壤氮素转化快,施氮处理施肥后第1～2天氨挥发出现峰值,氨挥发峰值为0.26～2.02 kg N·hm^-2·d^-1,7 d左右各处理氨挥发通量相近;施氮处理间氨累积排放量无显著差异;相同施氮量条件下,降低灌溉量氨累积排放量两季平均增加了46.7%;不同种植季氨平均排放通量和累积排放量均表现为西瓜季高于番茄季,西瓜季高温促进了氨排放;土壤铵态氮含量、土壤孔隙含水量、0～5 cm地温和温室气温均对氨排放通量有极显著影响,而土壤pH值与氨挥发通量呈显著负相关关系.不同种植季氨挥发通量和累积排放量存在差异,降低施氮量可减少氨排放,相同施氮量条件下降低灌溉量增加了氨排放.     

Electron paramagnetic resonance Signal II in spinach chloroplasts. I. Kinetic analysis for untreated chloroplasts

An analysis of electron paramagnetic resonance Signal II in spinach chloroplasts has been made using both continuous and flashing light techniques. In order to perform the experiments we developed a method which allows us to obtain fresh, untreated chloroplasts with low dark levels of Signal II. Under these conditions a single 10-μs flash is sufficient to generate greater than 80% of the possible light-induced increase in Signal II spin concentration. The risetime for this flash-induced increase in Signal II is approx. 1 s. The close association of Signal II with Photo-system II is confirmed by the observations that red light is more effective than is far red light in generating Signal II, and that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) does not inhibit the formation of the radical. Single flash saturation curves for the flash-induced increase in Signal I and Signal II indicate that the quantum efficiency for Signal II formation is close to that for Signal I. While one or two flashes (spaced 10 ms apart) are quite efficient in generating Signal II, three or four flashes are much less effective. However, if this spacing is decreased to 100 μs, three or four flashes become as efficient as one or two flashes. From observations of a deficiency of O₂ evolved during the initial flashes of dark-adapted chloroplasts, we conclude that the species which gives rise to Signal II is able to compete with water for oxidizing equivalents generated by Photosystem II. On the basis of these results we postulate a model in which Signal II arises from an oxidized radical which is produced by a slow electron transfer to the specific states S₂ and S₃ on the water side of Photo-system II.     

Improved solubility of β-cyclodextrin inclusion complexes by using liquid ammonia as a solvent and the possibility of asymmetric reduction

Dramatic improvement in the poor solubility of β-cyclodextrin (β-CD) and its inclusion complexes in water was achieved by using liquid ammonia (liq. NH₃) instead of water as the solvent. Asymmetric NaBH₄ reduction of the carbonyl groups of the inclusion complexes in liq. NH₃ was examined in a homogeneous condition to give the corresponding alcohols with moderate chirality.     

The reactions of zirconium(IV) bromide and zirconium(III) bromide and chloride with ammonia and zirconium(IV) bromide with ammonium cyanide

Unlike ZrCl₄, ZrBr₄ is not ammonolysed in liquid ammonia at temperatures up to −33 °C. The existence of ammoniates ZrBr₄nH₃ (n = 17, 12 and 9) at −36 °C has been established; at room temperature, the hexammine ZrBr₄ · 6NH₃ is the stable species which becomes ZrBr₄ · 2NH₃ at 200 °C. When treated with an excess of NH₄CN in liquid ammonia, complete replacement of bromide ions by cyanide occurs to give an inseparable mixture of Zr(CN)₄ · 2NH₃ and NH₄Br. The chloride and bromide of zirconium(III) also undergo no ammonolysis in liquid ammonia; the ammoniates stable at room temperature are ZrCl₃ · 2.5NH₃ and ZrBr₃ · 6NH₃.     

Characterization of pancreatic somatostatin binding sites with a I-somatostatin 28 analog

Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S₂₈): ¹²⁵I-[Leu⁸, DTrp²²,Tyr²⁵] S₂₈. The binding was highly dependent on calcium ions. In 0.2 mM free Ca²⁺ medium, binding at 37°C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (K_D=0.05±0.01 nM, B_max=157±33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca²⁺ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA^1,2[AzaAla^4–5,DTrp⁸,Phe^12–13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using ¹²⁵I-[Leu⁸,DTrp²²,Tyr²⁵]S₂₈ as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand ¹²⁵I-[Tyr¹¹]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors.     

The deactivation of Photosystem II in Chlorella as a second-order process

The kinetics of deactivation of the S₃ state in Chlorella have been observed under a variety of conditions. The S₃ state appears to decline in a dark period coming after a sequence of 30 saturating flashes in a second-order reaction, the rate constant of which is 0.132/[S*₃] s⁻¹ and which involves an electron donor, D₁, of concentration 1.25[S*₃] where [S*₃] is the concentration of the S₃ state when the oxygen yield of the light flashes is constant. If a 1 min period of 650 nm illumination is employed after the sequence of flashes, the subsequent S₃ state deactivation kinetics are more complex. There is an initial phase of S₃ state deactivation, accounting for about 35% of the original S₃ state, which is complete in less than 100 ms. The remaining 65% of the S₃ state appears to deactivate in a second-order reaction, the rate constant of which is 1.36/[S*₃] s⁻¹ and which involves an electron donor of initial concentration 0.58[S*₃]. If a 1 min period of 710 nm illumination comes after the 30 flashes, at least 98% of the S₃ state deactivates according to first-order kinetics. It is shown that this can be explained using a second-order model if there is an electron donor present of which the concentration is large compared with [S*₃]. However, S₃ state deactivation observed after 5 min of dark and two saturating flashes can be described neither by a first-order model nor a second-order model. Deactivation of the S₂ state after a 5 min dark period and one saturating flash follows second-order kinetics with a rate constant of 0.2/[S*₃] s⁻¹ and appears to involve an electron donor of initial concentration 1.3[S*₃]. Arguments are presented which tend to rule out the primary electron acceptor to Photosystem II as being any of the electron donors but it appears quite possible that the large plastoquinone pool is involved.     

Photooxidation of cytochrome b559 and the electron donors in chloroplast Photosystem II

The effect of light on the reaction center of Photosystem II was studied by differential absorption spectroscopy in spinach chloroplasts.

At − 196 °C, continuous illumination results in a parallel reduction of C-550 and oxidation of cytochrome b₅₅₉ high potential. With flash excitation, C-550 is reduced, but only a small fraction of cytochrome b₅₅₉ is oxidized. The specific effect of flash illumination is suppressed if the chloroplasts are preilluminated by one flash at 0 °C.

At − 50 °C, continuous illumination results in the reduction of C-550 but little oxidation of cytochrome b₅₅₉. However, complete oxidation is obtained if the chloroplasts have been preilluminated by one flash at 0 °C. The effect of preillumination is not observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

A model is discussed for the reaction center, with two electron donors, cytochrome b₅₅₉ and Z, acting in competition. Their respective efficiency is dependent on temperature and on their states of oxidation. The specific effect of flash excitation is attributed to a two-photon reaction, possibly based on energy-trapping properties of the oxidized trap chlorophyll.     

A low-temperature-sensitive intermediate state between S2 and S3 in photosynthetic water oxidation deduced by means of thermoluminescence measurements

The temperature dependence of S-state transitions in Photosystem II was measured by means of thermoluminescence using two different protocols for low-temperature flash excitation: protocol A, “last flash at low temperature”, and protocol B, “all flashes at low temperature”. Comparison of the temperature-dependence curves obtained by these two protocols revealed a marked difference particular for the three-flash experiments. The difference was attributed to the formation of a low-temperature sensitive precursor state between S₂ and S₃. The state is formed by two flash illumination given at −5 to −50°C, spontaneously transforms to normal S₃ on dark warming, and is not converted to S₀ by the 3rd flash. The precursor state was tentatively assigned to an S₃ in which H⁺ release is not completed.     

Structural organization of the oxidizing side of photosystem II. Exogenous reductants reduce and destroy the Mn-complex in photosystems II membranes depleted of the 17 and 23 kDa polypeptides

Removal of 23 and 17 kDa water-soluble polypeptides from PS II membranes causes a marked decrease in oxygen-evolution activity, exposes the oxidizing side of PS II to exogenous reductants (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) Biochim. Biophys. Acta 765, 388–398) and alters a high-affinity binding site for Ca²⁺ in the oxygen-evolving complex (Ghanotakis, D.F., Topper, J.N., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 170, 169–173). We have examined further the state of the functional Mn complex in PS II membranes from which the 17 and 23 kDa species have been removed by high-salt treatment. These membranes contain a structurally altered Mn complex which is sensitive to destruction by low concentrations of NH₂OH which cannot, in native PS II membranes, cause extraction of functional Mn. In addition to NH₂OH, a wide range of other small (H₂O₂, NH₂NH₂, Fe²⁺) and bulky (benzidine, hydroquinone) electron donors extract Mn (up to 80%) from the polypeptide-depleted PS II preparations. This extraction is due to reduction of the functional Mn complex since light, which would generate higher oxidation states within the Mn complex, prevents Mn release by reductants. Release of Mn by reductants does not extract the 33 kDa water-soluble protein implicated in Mn binding to the oxidizing side of PS II, although the protein can be partially or totally extracted from Mn-depleted preparations by exposure to high ionic strength or to high (0.8 M) concentrations of Tris. We view our results as evidence for a shield around the Mn complex of the oxygen-evolving complex comprised of the 33 kDa polypeptide along with the 23 and 17 kDa proteins and tightly bound Ca²⁺.     

Water proton relaxation as a monitor of membrane-bound manganese in spinach chloroplasts

First measurements of proton relaxation on chloroplast membranes are presented here. Experiments show that the water proton spin-lattice relaxation rate in chloroplast thylakoid membrane suspensions can be used to monitor membrane-bound manganese. The relaxation effect is reduced to 0.4 of its original value upon manganese extraction by washing with either alkaline Tris buffer or NH₂OH/EDTA solution. Large increases in the proton relaxation rate are measured in the presence of reductants such as tetraphenylboron and NH₂OH; oxidants such as potassium ferricyanide or 2,6-dichlorophenolindo-phenol lead to a decrease in this rate. These results suggest that maganese exists as a mixture of oxidation states in dark-adapted chloroplasts.