Kinetics of manganese redox transitions in the oxygen-evolving apparatus of photosynthesis

The kinetics of the S-state transitions of the oxygen-evolving complex were analyzed in dark-adapted, oxygen-evolving Photosystem-II preparations supplied with the electron acceptor 2,5-dichloro-p-benzoquinone. The kinetics of flash-induced absorbance changes at 350 nm, largely due to the successive S-state transitions S₀ → S₁, S₁ → S₂, S₂ → S₃ and S₃ →; S₀, confirm the +1, +1, +1, ?3 sequence of manganese oxidation reported earlier (Dekker, J.P., Van Gorkom, H.J., Wensink, J. and Ouwehand, L. (1984) Biochim. Biophys. Acta 767, 1–9), and reveal half-times of 30, 110, 350 and 1300 μs, respectively, for these transitions.     

The effects of mono- and divalent salts on the O2-evolution activity and low temperature multiline EPR spectrum of Photosystem II preparations from spinach

The ability of salts to inhibit the O₂-evolution activity of PS II preparations is shown to parallel closely the Hofmeister series, suggesting that inhibition is related to the solubility of the 16, 24 and 33 kDa proteins in these salt solutions. An examination of the effect of salt inactivation on the low temperature multiline EPR signal indicates that the release of either the 16 and 24 kDa proteins, or additionally the 33 kDa protein blocks or greatly reduces the efficiency of the advancement of the water-splitting complex to the S₂-state; under some conditions, this inhibition is reversible.     

Partial disintegration and reconstitution of the photosynthetic oxygen evolution system. Binding of 24 kilodalton and 18 kilodalton polypeptides

Treatment with 1 M NaCl almost totally removed two polypeptides of 24 and 18 kDa from the Photosystem II particles of spinach chloroplasts and reduced the oxygen-evolution activity by about half. Both polypeptides were able to rebind to the NaCl-treated particles in a low-salt medium. The rebinding of the 24 kDa polypeptide showed a saturation curve whose maximum level was close to that naturally occurring in the untreated particles. In parallel with the amount of rebound 24 kDa polypeptide, the oxygen-evolution activity was recovered. The 18 kDa polypeptide bound to the NaCl-treated particles without saturation. When the 18 kDa polypeptide was added to the particles previously treated with NaCl and then supplemented with a saturating amount of 24 kDa polypeptide, there appeared, in addition to the binding without saturation, another binding of the 18 kDa polypeptide with saturation to a maximum level close to that naturally occurring in the untreated particles. The 18 kDa polypeptide did not restore the oxygen-evolution activity. These findings suggest that there are specific binding sites; one for the 24 kDa polypeptide located on the Photosystem II particles, and the other for the 18 kDa polypeptide on the 24 kDa polypeptide.     

Assignment of the g = 4.1 ERP signal to manganese in the S2 state of the photosynthetic oxygen-evolving complex: An X-ray absorption edge spectroscopy study

X-ray absorption spectroscopy at the Mn K-edge has been utilized to study the origin of the g = 4.1 EPR signal associated with the Mn-containing photosynthetic O₂-evolving complex. Formation of the g = 4.1 signal by illumination of Photosystem II preparations at 140 K is associated with a shift of the Mn edge inflection point to higher energy. This shift is similar to that observed upon formation of the S₂ multiline EPR signal by 190 K illumination. The g = 4.1 signal is assigned to the Mn complex in the S₂ state.     

Absorbance difference spectra of the successive redox states of the oxygen-evolving apparatus of photosynthesis

The spectra of the absorbance changes due to the turnover of the so-called S-states of the oxygen-evolving apparatus were determined. The changes were induced by a series of saturating flashes in dark-adapted Photosystem II preparations, isolated from spinach chloroplasts. The electron acceptor was 2,5-dichloro-p-benzoquinone. The fraction of System II centers involved in each S-state transition on each flash was calculated from the oscillation pattern of the 1 ms absorbance transient which accompanies oxygen release. The difference spectrum associated with each S-state transition was then calculated from the observed flash-induced difference spectra. The spectra were found to contain a contribution by electron transfer at the acceptor side, which oscillated during the flash series approximately with a periodicity of two and was apparently modulated to some extent by the redox state of the donor side. At the donor side, the S₀ → S₁, S₁ → S₂ and S₂ → S₃ transitions were all three accompanied by the same absorbance difference spectrum, attributed previously to an oxidation of Mn(III) to Mn(IV) (Dekker, J.P., Van Gorkom, H.J., Brok, M. and Ouwehand, L. (1984) Biochim. Biophys. Acta 764, 301–309). It is concluded that each of these S-state transitions involves the oxidation of an Mn(III) to Mn(IV). The spectrum and amplitude of the millisecond transient were in agreement with its assignment to the reduction of the oxidized secondary donor Z⁺ and the three Mn(IV) ions.     

Calcium ions can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution

Photosystem II particles were prepared from spinach chloroplasts with Triton X-100, and treated with 1.0 M NaCl to remove polypeptides of 24 kDa and 18 kDa and to reduce the photosynthetic oxygen-evolution activity by about half. Oxygen-evolution activity was restored almost to the original level with 10 mM Ca²⁺, in a similar manner to the rebinding of 24-kDa polypeptide. Other cations such as magnesium, sodium and manganese ions could not restore any oxygen-evolution activity. These observations, together with a kinetic analysis, suggest that Ca²⁺ can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution in Photosystem II particles.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

Partial reconstitution of the photosynthetic oxygen evolution system by rebinding of the 33-kDa polypeptide

Treatment with 2.6 M urea of the Photosystem II particles depleted of two polypeptides of 24 kDa and 18 kDa completely released a polypeptide of 33 kDa and eliminated the oxygen-evolution activity. The 33-kDa polypeptide rebound to the urea-treated particles and partially reactivated the oxygen evolution. A quantitative analysis of the rebinding suggests tha there is a specific binding site for the 33-kDa polypeptide on the membrane surface.     

An improved purification method and a further characterization of the 33-kilodalton protein of spinach chloroplasts

The 33-kDa protein was purified in a high yield from thylakoid membranes of spinach chloroplasts. The extinction coefficient and A^1%_1cm value at 276 nm of the protein were 22000 M^?1·cm^?1 and 6.8, respectively. The 33-kDa protein and a polypeptide appearing at 32 kDa in the SDS-polyacrylamide gel electrophoresis of thylakoid membranes were compared by peptide mapping after limited proteolysis. This indicates that the 32-kDa band is entirely due to the 33-kDa protein. The molar ratio of chlorophyll to the 33-kDa protein in the chloroplasts was estimated to be 300. This suggests that one photosynthetic unit possesses one or two molecules of the 33-kDa protein.     

Studies on manganese binding by selective solubilization of Photosystem-II polypeptides

Deoxycholate was used to solubilize the 16 and 24 kDa polypeptides from spinach thylakoids, resulting in the loss of oxygen evolution. Manganese was retained in the membrane. When the deoxycholate-extracted membranes were subjected to a mild heat treatment, the water-soluble 33 kDa protein was selectively released. Less than one manganese per reaction center was lost on heating but this loss was not correlated to the solubilization of protein. Most of the manganese bound to the membrane remained EPR-undetectable and could be released by 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) or hydroxylamine treatments. This indicates that the manganese involved in oxygen evolution remains in its native binding site despite the loss of the 33 kDa protein. These results contradict the hypothesis that the 33 kDa protein is responsible for manganese binding at the photosynthetic oxygen-evolving site.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

The effect of temperature on the formation and decay of the multiline EPR signal species associated with photosynthetic oxygen evolution

We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S₂ state gives rise to this multiline signal; we find that the S₁ state can be fully advanced to the S₂ state at temperatures as low as 160 K. The S₂ state is capable of further advancement at temperatures above about 210 K, but not below that temperature.     

Effect of temperature on the photoreduction of centres A and B in Photosystem I,and the kinetics of recombination

When spinach Photosystem I particles, frozen in the dark with ascorbate, are illuminated at low temperatures, one electron is transferred from P-700 to either iron-sulphur centre A or B. It was found that the proportion of centre A or B reduced depended on the temperature of illumination. At 25 K, reduction of centre A, as detected by ESR spectroscopy, was strongly preferred. At higher temperatures, at about 150K, there was an increased proportion of reduced centre B. Reduction of B was more strongly preferred in particles frozen in 50% glycerol. The kinetics of dark reoxidation of A^? and B^? at various temperatures were followed by observing the radical signal of P-700⁺, and also by periodically cooling to 25 K to measure the ESR spectra of the iron-sulphur centres. The recombination of A^? and P-700⁺ occurred at lower temperatures than that at of B^?; at 150–200 K, centre B was the more stable electron trap. Dark reoxidation of both centres was more rapid in samples that were illuminated at 25 K than in samples illuminated at 150–215 K. In no case was net electron transfer between centres A and B observed. Differences in g values of the ESR spectra in particles illuminated at 25 and 200 K indicate that the iron-sulphur centres are in altered conformational states. It is concluded firstly that, in the frozen state, the rates of dark electron transfer decrease in the sequence A^? → P-700⁺ > B^? → P-700⁺ > B^? → A; secondly, that when centres A or B are photoreduced, a temperature-dependent conformational change takes place which slows down the rate of recombination with P-700⁺.     

EPR studies of the oxygen-evolving enzyme of Photosystem II

The light-induced EPR multiline signal is studied in O₂-evolving PS II membranes. The following results are reported: (1) Its amplitude is shown to oscillate with a period of 4, with respect to the number of flashes given at room temperature (maxima on the first and fifth flashes). (2) Glycerol enhances the signal intensity. This effect is shown to come from changes in relaxation properties rather than an increase in spin concentration. (3) Deactivation experiments clearly indicate an association with the S₂ state of the water-oxidizing enzyme. A signal at g = 4.1 with a linewidth of 360 G is also reported and it is suggested that this arises from an intermediate donor between the S states and the reaction centre. This suggestion is based on the following observations: (1) The g = 4.1 signal is formed by illumination at 200 K and not by flash excitation at room temperature, suggesting that it arises from an intermediate unstable under physiological conditions. (2) The formation of the g = 4.1 signal at 200 K does not occur in the presence of DCMU, indicating that more than one turnover is required for its maximum formation. (3) The g = 4.1 signal decreases in the dark at 220 K probably by recombination with Q^?_AFe. This recombination occurs before the multiline signal decreases, indicating that the g = 4.1 species is less stable than S₂. (4) At short times, the decay of the g = 4.1 signal corresponds with a slight increase in the multiline S₂ signal, suggesting that the loss of the g = 4.1 signal results in the disappearance of a magnetic interaction which diminishes the multiline signal intensity. (5) Tris-washed PS II membranes illuminated at 200 K do not exhibit the signal.     

Role of the 33-kDa polypeptide in preserving Mn in the photosynthetic oxygen-evolution system and its replacement by chloride ions

Treatment of Photosystem II particles from spinach chloroplasts with Triton X-100 with 2.6 M urea in the presence of 200 mM NaCl removed 3 polypeptides of 33 kDa, 24 kDa and 18 kDa, but left Mn bound to the particles. The (urea + NaCl)-treated particles could evolve oxygen in 200 mM, but not in 10 mM NaCl. Mn was gradually released with concomitant loss of oxygen-evolution activity in 10 mM NaCl but not in 200 mM Cl^?. The NaCl-treated particles, which contained Mn and the 33-kDa polypeptide but not the 24-kDa and 18-kDa polypeptides, did not lose Mn or oxygen-evolution activity in 10 mM NaCl. These observations suggest that the 33-kDa polypeptide maintains the binding of Mn to the oxygen-evolution system and can be functionally replaced by 200 mM Cl^?.     

Purification of cytochrome b-559 from oxygen-evolving Photosystem II preparations of spinach and maize

A rapid and simple procedure is presented for the purification of chloroplast cytochrome b-559. The method is based on the protocol devised by Garewal and Wasserman (Garewal, H.S. and Wasserman, A.R. (1974) Biochemistry 13, 4063–4071), which we have modified to eliminate the requirement for a lengthy electrophoretic step. Novel features of our method include: the use of oxygen-evolving Photosystem II preparations (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539) as the starting material; isocratic elution of cytochrome b-559 from a DEAE-cellulose column (yielding the protein in a pure state); and a simple column procedure for removal of excess Triton X-100. The procedure has been applied to both spinach and maize (Zea mays L.). Purified cytochromes b-559 from these species have similar optical spectra and mobility during gel electrophoresis under native conditions. Lithium dodecyl sulfate polyacrylamide gel electrophoresis of cytochrome b-559 from both spinach and maize reveals a major polypeptide band (apparent molecular mass = 9 kDa), and two minor bands (apparent molecular masses = 10 kDa and 6 kDa).     

Regulation of Photosystem I electron flow activity by phosphatidylglycerol in thylakoid membranes as revealed by phospholipase treatment

Thylakoid membranes were treated by potato lipolytic acyl hydrolase, phospholipases A₂ from pancreas and snake venom, and by phospholipase C from Bacillus cereus under various conditions. The changes in the uncoupled rates of electron transport through Photosystem I (PS I) and in lipid composition were followed during these treatments. Pancreatic phospholipase A₂ which destroyed all phospholipids in thylakoid membranes stimulated the NADP⁺ reduction supported by reduced 2,6-dichlorophenolindophenol. This stimulation concerned only the dark but not the light reactions of this pathway. The main site of action of pancreatic phospholipase A₂ may be located on the donor side of PS I; the hydrolysis of phospholipids at this site caused an increased ability of reduced 2,6-dichlorophenolindophenol and ascorbate alone to feed electrons into PS I. A second site may be located on the acceptor side of PS I, probably between the primary acceptor and the ferredoxin system. When thylakoid membranes were first preincubated with or without lipolytic acyl hydrolase at 30°C (pH 8), the NADP⁺ photoreduction was inhibited whilst the methyl viologen-mediated O₂ uptake was stimulated. A subsequent addition of pancreatic phospholipase A₂ (which had the same hydrolysis rates for phosphatidylglycerol but not for phosphatidylcholine) further stimulated the O₂ uptake and restored NADP⁺ photoreduction. The extent of this stimulation, which depended on the presence of lipolytic acyl hydrolase, was ascribed partly to the hydrolysis of the phospholipids and partly to the generation of their lyso derivatives but not to the release of free fatty acids. On the contrary, phospholipase C which destroyed only phosphatidylcholine failed to restore this activity. It is suggested that phosphatidylglycerol is the only phospholipid associated with thylakoid membrane structures supporting PS I activities and that this lipid may play a physiological role in the regulation of these activities.     

Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution: II. A binuclear manganese-containing 34 kilodalton protein,a probable component of the water dehydrogenase enzyme

Extraction conditions have been found which result in the retention of managanese to the 33–34 kDa protein, first isolated as an apoprotein by Kuwabara and Murata (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys Acta 581, 228–236). By maintaining an oxidizing-solution potential, with hydrophilic and lipophilic redox buffers during protein extraction of spinach grana-thylakoid membranes, the 33–34 kDa protein is observed to bind a maximum of 2 Mn/protein which are not released by extended dialysis versus buffer. This manganese is a part of the pool of 4 Mn/Photosystem II normally associated with the oxygen-evolving complex. The mechanism for retention of Mn to the protein during isolation appears to be by suppression of chemical reduction of natively bound, high-valent Mn to the labile Mn(II) oxidation state. This protein is also present in stoichiometric levels in highly active, O₂-evolving, detergent-extracted PS-II particles which contain 4–5 Mn/PS II. Conditions which result in the loss of Mn and O₂ evolution activity from functional membranes, such as incubation in 1.5 mM NH₂OH or in ascorbate plus dithionite, also release Mn from the protein. The protein exists as a monomer of 33 kDa by gel filtration and 34 kDa by gel electrophoresis, with an isoelectric point of 5.1 ± 0.1. The protein exhibits an EPR spectrum only below 12 K which extends over at least 2000 G centered at g = 2 consisting of non-uniformly separated hyperfine transitions with average splitting of 45–55 G. The magnitude of this splitting is nominally one-half the splitting observed in monomeric manganese complexes having O or N donor ligands. This is apparently due to electronic coupling of the two ⁵⁵Mn nuclei in a presumed binuclear site. Either a ferromagnetically coupled binuclear Mn₂(III,III) site or an antiferromagnetically coupled mixed-valence Mn₂(II,III) site are considered as possible oxidation states to account for the EPR spectrum. Qualitatively similar hyperfine structure splittings are observed in ferromagnetically coupled binuclear Mn complexes having even-spin ground states. The extreme temperature dependence suggests the population of low-lying excited spin states such as are present in weakly coupled dimers and higher clusters of Mn ions, or, possibly, from efficient spin relaxation such as occurs in the Mn(III) oxidation state. Either 1.5 mM NH₂OH or incubation with reducing agents abolishes the low temperature EPR signal and releases two Mn(II) ions to solution. This is consistent with the presence of Mn(III) in the isolated protein. The intrinsically unstable Mn₂(II,III) oxidation state observed in model compounds favors the assignment of the stable protein oxidation state to the Mn₂(III,III) formulation. This protein exhibits characteristics consistent with an identification with the long-sought Mn site for photosynthetic O₂ evolution. An EPR spectrum having qualitatively similar features is observable in dark-adapted intact, photosynthetic membranes (Dismukes, G.C., Abramowicz, D.A., Ferris, F.K., Mathur, P., Upadrashta, B. and Watnick, P. (1983) in The Oxygen-Evolving System of Plant Photosynthesis (Inoue, Y., ed.), pp. 145–158, Academic Press, Tokyo) and in detergent-extracted, O₂-evolving Photosystem-II particles (Abramowicz, D.A., Raab, T.K. and Dismukes, G.C. (1984) Proceedings of the Sixth International Congress on Photosynthesis (Sybesma, C., ed.), Vol. I, pp. 349–354, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands), thus establishing a direct link with the O₂ evolving complex.     

Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites in Photosystem II of spinach chloroplasts

Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites were found in PS II of spinach chloroplasts, depending on the pH of the assay medium used. The low site (pH 6) can be inhibited by certain quinolines, such as 8-hydroxyquinoline at concentrations less than 50 μM. The high pH site (pH 8) can be inhibited by disodium cyanamide, folic acid, or 5,6-benzoquinoline at concentrations from 50 μM to 5 mM. With the exception of orthophenanthroline, which stimulates the high pH site but does not show much inhibition at low pH, all other inhibitors gave opposite effects at the pH values used, i.e., they stimulated at low pH or inhibited at high pH, or vice versa. Several mechanisms for the observed effects are discussed.     

Oxygen-evolution patterns from spinach Photosystem II preparations

Patterns of O₂ evolution resulting from sequences of short flashes are reported for Photosystem (PS) II preparations isolated from spinach and containing an active, O₂-evolving system. The results can be interpreted in terms of the S-state model developed to explain the process of photosynthetic water splitting in chloroplasts and algae. The PS II samples display damped, oscillating patterns of O₂ evolution with a period of four flashes. Unlike chloroplasts, the flash yields of the preparations decay with increasing flash number due to the limited plastoquinone acceptor pool on the reducing side of PS II. The optimal pH for O₂ evolution in this system (pH 5.5–6.5) is more acidic than in chloroplasts (pH 6.5–8.0). The O₂-evolution, inactivation half-time of dark-adapted preparations was 91 min (on the rate electrode) at room temperature. Dark-inactivation half-times of 14 h were observed if the samples were aged off the electrode at room temperature. Under our conditions (experimental conditions can influence flash-sequence results), deactivation of S₃ was first order with a half-time of 105 s while that of S₂ was biphasic. The half-times for the first-order rapid phase were 17 s (one preflash) and 23 s (two preflashes). The longer S₂ phase deactivated very slowly (the minimum half-time observed was 265 s). These results indicate that deactivation from S₃ → S₂ → S₁, thought to be the dominant pathway in chloroplasts, is not the case for PS II preparations. Finally, it was demonstrated that the ratio of S₁ to S₀ can be set by previously developed techniques, that S₀ is formed mostly from activated S₃ (S₄), and that both S₀ and S₁ are stable in the dark.