Reduction kinetics of the photo-oxidized chlorophyll a+II in the nanosecond range: Measurements of the absorption changes at 688 nm under repetitive flash excitation

The 688 nm absorption changes (ΔA₆₈₈), indicating the photochemical turnover of chlorophyll a_II (Chl a_II) have been investigated under repetitive laser flash excitation conditions in spinach chlorplasts. It was found that under steady state conditions about 50–60% of the photo-oxidized primary donor of Photosystem II (PS II), Chl a⁺_II, becomes re-reduced with a biphasic kinetics in the nanosecond time scale with half-life times of about 50 ns and 400 ns. The remaining Chl a⁺_II becomes re-reduced in the microsecond range.     

Reversible alteration of nanosecond reduction of chlorophyll a+II in inside-out thylakoids correlated to inhibition and reconstitution of oxygen-evolving activity

The electron donation to Chl a⁺_II has been studied by measurement of absorbance changes at 824 nm under repetitive excitation conditions. For untreated inside-out thylakoids the electron donation was dominated by 35 and 220 ns kinetics. After salt-washing, both oxygen-evolution and nanosecond phases decreased drastically with corresponding increase in the microsecond time range. On addition of a purified 23 kDa protein, a restoration of the nanosecond phases up to 75% of the orginal level was obtained concomitant with a corresponding restoration of oxygen evolution. The results are consistent with a function of the 23 kDa protein at the oxidizing side of Photosystem II and that the nanosecond donation to Chl-a⁺_II is coupled to the natural path of electrons from water.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Depletion of Photosystem II-extrinsic proteins. I. Effects on O2- and N2-flash yields and steady-state O2 evolution

Wheat O₂-evolving Photosystem II (PS II) membranes having a PS II unit of approx. 200 chlorophylls (Chl), approx. 4 Mn/200 Chl, less than 1 P-700/3000 Chl and an electron-acceptor pool of approx. 2.5 equiv./PS II were analyzed and compared with wheat PS II membranes depleted (at least 90%) of the 17 and 23 kDa proteins by NaCl extraction during Triton X-100 isolation of membranes. Extraction of these proteins caused approx. 50% decrease in O₂ evolution in any light regime and an increase of approx. 2 equiv./PS II of the electron-acceptor pool, but affected neither Mn abundance, photoreduction of DCIP by tetraphenylboron, or N₂ yield (from NH₂OH) from a single flash. Mass spectrometric analyses of O₂ flash yields in the presence of potassium ferricyanide showed that both chloroplasts and the unextracted PS II membranes yielded oscillations compatible with S₀/S₁/S₂/S₃ of 25:75:0:0 and α (0.1) and β (0.05). Depletion of 17 and 23 kDa proteins resulted in a two-fold increase in α, approx. 25–40% disconnection of the S state complex from the PS II trap complex but with no change in β. Preincubation of control or extracted PS II membranes with potassium ferricyanide permitted a significant double-hit on the first flash. In the absence of an added electron acceptor, N₂ flash yields were more sustained with 17 and 23 kDa depleted than with 17 and 23 kDa sufficient PS II membranes. In contrast, no significant O₂ flash yields were observed with extracted PS II preparations under these conditions (control PS II membranes showed a predictable O₂ pattern before damping after only 5–6 flashes). These results suggest that extraction of the 17 and 23 kDa proteins results in an increase of pool size on the PS II acceptor side (seen as unmasking ‘Component C’). ‘Component C’ can mediate electron transfer from Q⁻ to Z⁺ (S₂).     

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a_II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a_II and X320 unless exogenous acceptors are present, which directly oxidize X320^?, such as K₃Fe(CN)₆. (c) In the presence of K₃Fe(CN)₆ the recovery kinetics of Chl a_II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S₂ and S₃ in normal chloroplasts, significantly accelerates the recovery of Chl a_II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D₁ to Chl a⁺_II. ANT 2p simultaneously retards the decay kinetics of X320^? and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320^? and Chl a⁺_II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D⁺₁ between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.     

Total recovery of O2 evolution and nanosecond reduction kinetics of chlorophyll-a II + (P-680+) after inhibition of water cleavage with acetate

Oxygen evolution and reduction kinetics of the photooxidized Chl-a_II ⁺ have been measured in oxygen-evolving complexes from the thermophilic cyanobacterium Synechococcus sp.

1. Incubation of PS II particles with acetate resulted in an inhibition of oxygen evolution and a retardation of the Chl-a_II ⁺=reduction kinetics from the nanosecond range to the microsecond range, indicating a modification of the donor side of photosystem II (PS II).
2. After the first two flashes given to a dark-adapted, acetate treated sample, Chl-a_II ⁺ was re-reduced with a half-life time of 160 μs by a component of the donor side of PS II. Under repetitive excitation Chl-a_II ⁺ was re-reduced in 500 μs by electron back reaction from the primary acceptor Q_A ^- (X-320^-). Obviously, in the presence of acetate only two electrons are available from the donor side.
3. Both oxygen evolution and nanosecond reduction kinetics of Chl-a_II ⁺ were restored to the control level when acetate was removed.
4. The results indicate a tight coupling between O₂ evolution and nanosecond reduction kinetics of Chl-a_II ⁺.
5. The reversible inhibition is probably due to a replacement of Cl^- by acetate within the water splitting enzyme.
6. Due to its strongly retarded kinetics, the reversibly modified system may facilitate investigations of the mechanism of the donor side.

     

Some properties of Photosystem II preparations from the cyanobacterium Synechococcus sp. — presence of an l-amino acid oxidase in Photosystem II complexes from Synechococcus sp.

A highly active O₂-evolving Photosystem II complex has been purified from the cyanobacterium Synechococcus sp., and this complex has been compared with the Photosystem II complex previously isolated from this cyanobacterium (Ohno, T., Satoh, K. and Katoh, S. (1986) Biochim. Biophys. Acta 852, 1–8). Further treatment of the O₂-evolving complex with the detergent sodium taurodesoxycholate resulted in a complex which consisted mainly of the 47 and 40 kDa peptides and which had lost the O₂-evolving activity, but which could still reduce 2,6-dichlorophenolindophenol with 1,5-diphenylcarbazide. Previously, we have shown that a flavoprotein of 49 kDa which has an l-amino acid oxidase activity under certain conditions, is a component of highly active Photosystem II preparations from the cyanobacterium Anacystis nidulans (Pistorius, E.K. and Gau, A.E. (1986) FEBS Lett. 206, 243–248). Based on immunological studies with the antiserum raised against the l-amino acid oxidase protein from A. nidulans, we show that a protein which cross-reacts with this antiserum is present in the highly purified Photosystem II preparations from Synechococcus sp. Moreover, an l-amino acid oxidase activity could also be detected in Photosystem II preparations from Synechococcus sp. The enzyme preferentially oxidizes basic l-amino acids as l-arginine, l-ornithine, 2,3-diamino propionic acid and l-citrulline. In contrast to the enzyme from A. nidulansl-lysine is not oxidized. The here shown presence of an l-amino acid oxidase protein in Photosystem II preparations from Synechococcus sp. is an additional support of our hypothesis that a flavoprotein is a functional component of the water-oxidizing enzyme complex.     

The state of manganese in the photosynthetic apparatus: 4. Structure of the manganese complex in Photosystem II studied using EXAFS spectroscopy. The S1 state of the O2-evolving Photosystem II complex from spinach

The structure of the Mn complex in the oxygen-evolving system and its mechanistic relation to photosynthetic oxygen evolution are poorly understood, though many studies have established that membrane-bound Mn plays an active role. Recently established procedures for isolating oxygen-evolving subchloroplast Photosystem II (PS II) preparations and the discovery of a light-induced multiline EPR signal attributable to the S₂ state of the O₂-evolving complex have facilitated the preparation of samples well characterized in the S₁ and S₂ states. We have used extended X-ray absorption fine structure (EXAFS) spectroscopy to probe the ligand environment of Mn in PS II particles from spinach, and in this report we present our results. The essential feature of the EXAFS results are that at least two Mn atoms per PS II reaction center occur as a binuclear species with a metal-metal distance of approx. 2.7 Å, with low Z atoms, N or O, at a distance of approx. 1.75 Å and at approx. 1.98 Å, which are characteristic of bridging and terminal ligands. These results agree well with those derived from whole chloroplasts that provided the first evidence for a binuclear manganese complex (Kirby, J.A., Robertson, A.S., Smith, J.P., Thompson, A.C., Cooper, S.R. and Klein, M.P. (1981) J. Am. Chem. Soc. 103, 5529–5537).     

Presence of a flavoprotein in O2-evolving Photosystem II preparations from the cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem II complex which was greatly depleted of phycobiliproteins was isolated from the cyanobacterium Anacystis nidulans. This complex contained the flavoprotein with l-amino acid oxidase activity which we have previously shown to be present in thylakoid preparations of this cyanobacterium (Pistorius, E.K. and Voss, H. (1982) Eur. J. Biochem. 126, 203–209). One of the most prominent polypeptides in this O₂-evolving Photosystem II complex had a molecular weight of 49 kDa. This polypeptide co-chromatographed on SDS-polyacrylamide gels with the purified l-amino acid oxidase which consists of two subunits of 49 kDa. The antagonistic effect of CaCl₂ on the two examined reactions could also be demonstrated with this O₂-evolving Photosystem II complex: CaCl₂ stimulated photosynthetic O₂ evolution, but inhibited the l-amino acid oxidase activity. Both reactions were inhibited by o-phenanthroline. These results further support a functional relationship between the flavoprotein with l-amino acid oxidase activity and Photosystem II activities in A. nidulans. However, we only found 1 mol FAD per 350–650 mol chlorophyll, although 1 gatom Mn per 5–10 mol chlorophyll was present. When we assume a photosynthetic unit of about 40 chlorophylls, then in most preparations the FAD values were more than a factor of 10 too low. Results which we obtained with the purified l-amino acid oxidase showed that the FAD values were in most enzyme samples lower than the theoretically expected value of 2 mol FAD per mol enzyme. Moreover, in some cases the absorption spectrum of the enzyme showed substantial deviations from the spectrum of oxidized FAD. These experiments indicated that the flavin in the enzyme could partly exist in a form which was different from ‘authentic oxidized FAD’. We do not yet know the chemical nature of this ‘modified flavin’.     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.     

Rise time of EPR signal IIvf in chloroplast Photosystem II

The rise time of the photoinduced, reversible EPR Signal II_vf in spinach chloroplasts is found using flash excitation to be 20 ± 10 μs. The results are interpreted as evidence that the Signal II_vf radical is an electron carrier on the donor side of Photosystem II, but probably does not result from the first donor to P680⁺.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Preparation of O2-evolving Photosystem-II subchloroplasts from spinach

An O₂-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O₂ evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O₂ uptake activity. The preparation showed a chlorophyll $\text{a}\text{b}$ ratio of 1.9 and a $\text{P-700}\text{chlorophyll}$ ratio of $\text{1}\text{2400}$. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O₂-evolving Photosystem II preparation.     

Development of Photosystem I and Photosystem II Activities in Leaves of Light-grown Maize (Zea mays)

To compare chloroplast development in a normally grown plant with etiochloroplast development, green maize plants (Zea mays), grown under a diurnal light regime (16-hour day) were harvested 7 days after sowing and chloroplast biogenesis within the leaf tissue was examined. Determination of total chlorophyll content, ratio of chlorophyll a to chlorophyll b, and O₂-evolving capacity were made for intact leaf tissue. Plastids at different stages of development were isolated and the electron-transporting capacities of photosystem I and photosystem II measured. Light saturation curves were produced for O₂-evolving capacity of intact leaf tissue and for photosystem I and photosystem II activities of isolated plastids. Structural studies were also made on the developing plastids. The results indicate that the light-harvesting apparatus becomes increasingly efficient during plastid development due to an increase in the photosynthetic unit size. Photosystem I development is completed before that of photosystem II. Increases in O₂-evolving capacity during plastid development can be correlated with increased thylakoid fusion. The pattern of photosynthetic membrane development in the light-grown maize plastids is similar to that found in greening etiochloroplasts.     

Studies on the water-oxidizing system by the effects of different treatments in chloroplasts

Treatments such as trypsinization (50 μg/ml per mg Chl for 1 h), osmotic shock of the chloroplasts or mild heating altered the oxygen evolution in such a way that the properties of the Photosystem II were simplified. After these treatments, the damping of the oscillation pattern of O₂ yields induced by a flash series remained the same, irrespective of the level of inhibition induced by the treatment. This damping did not decrease with increasing flash energy, as observed in untreated chloroplasts. The light saturation curve of the S₂ → S₃ transition of the O₂ evolving system no more exhibited the slow-increasing phase at high flash energy observed under normal conditions. The kinetic properties of the O₂-evolving system were also simplified. After the treatments cited above, deactivation of S₂ and S₃ were identical and accelerated with respect to untreated chloroplasts. Turnover kinetics of the transitions S₁ → S₂ and S₂ → S₃ were also similar and simpler without a lag for S₂ → S₃. These results indicate that the treatments mentioned above disconnect one donor from the O₂-evolving complex. This donor, under normal conditions, contributes to the increase of the quantum yield of the transition S₂ → S₃ at high flash energy. This donor is here denoted by D. Our results are in agreement with the following working hypothesis: the large miss, observed on the S₂ → S₃ transition without any contribution of the donor D, may be due to the fact that the system needs a conformation change of the O₂-evolving complex in the S₂ state, so that the main donor Y can oxidize the second H₂O molecule in the water-splitting complex. In the inactive state corresponding to the absence of a conformation change, the donor D, being different in configuration, is likely to oxidize the S₂ state into an S₃ state at high light intensity.     

Involvement of the donor tyrosine-D1 (Yd) in Photosystem II electron transport in the green alga, Chlamydobotrys stellata

The light-induced oxidation of the accessory donor tyrosine-D (Y_D) has been studied by measurements of the EPR Signal II_slow at room temperature in the autotrophically and photoheterotrophically cultivated alga Chlamydobotrys stellata. After illumination and dark adaptation, Y_D Signal II_slow was observed only in autotrophic algae, i.e. under conditions of a linear photosynthetic electron transfer from water to NADP⁺. The addition of artificial electron acceptors phenyl-p-benzoquinone (PPQ) or dichloro-p-benzoquinone (DCQ) to the autotrophic cells caused an almost negligible increase of this signal. When photosynthetic electron flow and oxygen evolution were diminished by removal of the carbon source CO₂ and addition of acetate (photoheterotrophy), a pronounced Y_D Signal II_slow was seen only in presence of DCQ or PPQ. Several possibilities are discussed to explain the absence of Y_D Signal II_slow in photoheterotrophic Chl. stellata such as the existence of a cyclic PS II electron flow very effectively reducing P₆₈₀ and thereby preventing the possibility of Y_D oxidation. Artificial electron acceptors withdraw electrons from this cycle thus keeping the primary quinone acceptor, Q_A, oxidized and thereby diminishing the reduction of P₆₈₀ ⁺ by cyclic PSII. This leads to the appearance of the Y_D Signal II_slow also in the photoheterotrophically grown algae.Abbreviations A-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - DCQ- 2,5-dichlorobenzoquinone - D₂- structure protein of Photosystem II - EPR- electron paramagnetic resonance - OEC- oxygen evolving complex - PPQ- phenyl-p-benzoquinone - PS II- Photosystem II - P₆₈₀- reaction center of Photosystem II - Q-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - S_i- oxidation levels of the OEC - Y_D- tyrosine-D accessory donor to P₆₈₀ - Y_Z- tyrosine-Z electron donor to P₆₈₀ Dedicated to Prof. Dr E. Schnepf/Heidelberg.     

Structural,biochemical and biophysical characterization of four oxygen-evolving Photosystem II preparations from spinach

Four procedures utilizing different detergent and salt conditions were used to isolate oxygen-evolving Photosystem II (PS II) preparations from spinach thylakoid membranes. These PS II preparations have been characterized by freeze-fracture electron microscopy, SDS-polyacrylamide gel electrophoresis, steady-state and pulsed oxygen evolution, 77 K fluorescence, and room-temperature electron paramagnetic resonance. All of the O₂-evolving PS II samples were found to be highly purified grana membrane fractions composed of paired, appressed membrane fragments. The lumenal surfaces of the membranes and thus the O₂-evolving enzyme complex, are directly exposed to the external environment. Biochemical and biophysical analyses indicated that all four preparations are enriched in the chlorophyll $\text{a}\text{b-}\text{light-harvesting}$ complex and Photosystem II, and depleted to varying degrees in the stroma-associated components, Photosystem I and the CF₁-ATPase. The four PS II samples also varied in their cytochrome f content. All preparations showed enhanced stability of oxygen production and oxygen-rate electrode activity compared to control thylakoids, apparently promoted by low concentrations of residual detergent in the PS II preparations. A model is presented which summarizes the effects of the salt and detergent treatments on thylakoid structure and, consequently, on the configuration and composition of the oxygen-evolving PS II samples.