The back reaction in the primary electron transfer couple of photosystem II of photosynthesis

Changes of C-550, cytochrome b₅₅₉ and fluorescence yield induced in chloroplasts by single saturating flashes were studied at low temperature. A single saturating flash at −196°C was quite ineffective in reducing C-550, oxidizing cytochrome b₅₅₉ or increasing the fluorescence yield, presumably because most of the charge separation induced by the flash was dissipated by a direct back reaction in the primary electron transfer couple. The back reaction, which competes with the dark reduction of the oxidized primary electron donor by a secondary electron donor, becomes increasingly important as the temperature is lowered because of the temperature coefficient of the reaction with the secondary donor. The effect of the back reaction is to lower the quantum yield for the production of stable photochemical products by steady irradiation. Assuming a quantum yield of unity for the photoreduction of C-550 at room temperature, the quantum yield for the reaction is about 0.40 at −100°C and 0.27 at −196°C.     

The 520 nm absorbance changes in Scenedesmus obliquus and its relation to Photosystem I

The kinetics (region of seconds) of the light-induced 520 nm absorbance change and its dark reversal have been studied in detail in the wild type and in some pigment and photosynthetic mutants of Scenedesmus obliquus. The following 5 lines of evidence led us to conclude that the signal is entirely due to the photosystem I reaction modified by electron flow from Photosystem II.Gradual blocking of the electron transport with 3(3,4-dichlorophenyl)-1,1-dimethylurea resulted in diminution and ultimate elimination of the biphasic nature of the signal without reducing the extent of the absorbance change or of the dark kinetics. On the contrary, blocking electron flow at the oxidizing side of plastoquinone with 2, $\text{5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isoprophyl}\text{-p-}\text{benzoquinone}$ or inactivating the plastocyanin with KCN, prolonged the dark reversal of the absorbance change apart from abolishing the biphasic nature of the signal.Action spectra clearly indicate that the main signal (I) is due to electron flow in Photosystem I and that its modification (Signal II) is due to the action of Photosystem II.Signal I is pH independent, whereas Signal II demonstrates a strong pH dependence, parallel to the O₂-evolving capacity of the cells.Chloroplast particles isolated from the wild type Scenedesmus cells demonstrated in the absence of any added artificial electron donor or acceptor and also under non-phosphorylation conditions the 520 nm absorbance change with approximately the same magnitude as whole cells. The dark kinetics of the particles were comparatively slower. Removal of plastocyanin and other electron carriers by washing with Triton X-100 slowed down the kinetics of the dark reversal reaction to a greater extent. A similar positive absorbance change at 520 nm and slow dark reversal was also observed in the Photosystem I particles prepared by the Triton method.Mutant C-6E, which contains neither carotenoids nor chlorophyll $\text{b}$ and lacks Photosystem II activity, demonstrates a normal signal I of the 520 nm absorbance change. This latter result contradicts the postulate that carotenoids are the possible cause of the 520 nm absorbance change.     

The I-D fluorescence transient. An indicator of rapid energy distribution changes in photosynthesis

The I-D transient in the chlorophyll fluorescence induction curve (Kautsky effect) is investigated in the view of recently discovered rapid changes in energy distribution between the two photosystems (Schreiber, U. and Vidaver, W., FEBS Lett., in the press). Fluorescence induction curves differ appreciably depending on whether measured at λ < 690 nm, originating in pigment system II, or at λ > 715 nm, which is in part from pigment system I. The differences occur as well in the rapid part of the induction curve (O-I-D-P) as in the slower P-S decay. Most significant changes in energy distribution are indicated in the region of the I-D dip, being induced by appropriate preillumination. The effect is studied by (a) comparing the individual fluorescence time courses at λ < 690 nm and λ > 715, (b) plotting F < 690 vs. F > 715 and (c) recording time courses of $\text{F < 690}\text{F > 715}$ ratios. In (a) the I and D characteristics are delayed at F > 715 relative to F < 690, which is accompanied by periods close to I and D, where the two emissions follow inverse courses. In (b) the I-D dip corresponds to a loop. And in (c) it is shown that a rapid ratio decay, reflecting increasing excitation of System I pigments, is initiated before the I-D dip. These data indicate that the I-D transient is caused by a rapid switch of energy distribution in favor of System I and the resulting stimulation of Q reoxidation via the electron transport chain. It is suggested that as with the slow fluorescence transients the rapid also can be understood as a composite of two different changes, (1) direct changes resulting from a switch in energy distribution, which are inverse for F < 690 and F > 715, and (2) indirect changes due to stimulated Q reduction or Q oxidation, which are parallel for both emissions. The rapid ratio decay, correlated to I-D, persists and is even stimulated in the presence of electron transport inhibitors. This and the speed of the phenomenon make it improbable that the rapid energy distribution changes are affected by an ion flux-induced mechanism. It is proposed that the electrical field across the thylakoid membrane is involved in the energy switch mechanism.     

Analysis of absorption changes in the ultraviolet related to charge-accumulating electron carriers in Photosystem II of chloroplasts

Spinach chloroplasts were dark adapted and then submitted to a sequence of short saturating flashes. The resulting absorption changes in the near ultraviolet were analyzed and attributed to the donor and acceptor sides of Photosystem II. Our results provide a spectroscopic support to some current models of these parts of the photosynthetic electron transport.In Tris-treated chloroplasts (supplied with artificial donors) the absorption changes are largely due to the acceptor side. After each flash the signal decays with a fast phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{ms at}\text{9 °}\text{C}$) leaving a stationary level (on a 100-ms time scale). The fast phase has a small amplitude after odd-numbered flashes, whereas the stationary level behaves in a complementary fashion. The non-decaying signal is attributed mostly to the reduced secondary acceptor (A₂^?) and the fast phase to the simultaneous reoxidation of A₂^? and of the reduced primary acceptor (A₁^?). The effect of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and of redox mediators (ascorbate, ferricyanide) also support this assignment. A fraction of A₂ is shown to be reduced in dark-adapted chloroplasts, as proposed by Velthuys and Amesz (Biochim. Biophys. Acta (1974) 333, 85–94). The difference spectra support the view that A₁^? and A₂^? are plastoquinone radical anions. There are also some absorption changes that we cannot identify.In untreated chloroplasts a non-decaying absorption change (“slow phase”) occurs with a 4-flash periodicity. It is attributed to the transitions among the S states associated with the O₂-evolving complex. A fast phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{ms}$) in the decay following the first two flashes behaves like in Tris-treated chloroplasts, so that the assignment is tentatively the same. After the third flash, however, the magnitude of this fast phase is too large according to the hypothesis, so that there may be some contribution from the donor side. The fast phases become slower at lower pH (5.5 instead of 7.6), although there is no evidence for a protonation A₁^? or A₂^?.     

Oxygen evolution in the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421

The oxygen-evolving reactions of the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421 were compared with those of Synechocystis sp. PCC 6803. Four aspects were considered: sequence conservation in three extrinsic proteins for oxygen evolution, steady-state oxygen-evolving activity, charge recombination reactions, i.e., thermoluminescence and oscillation patterns of delayed luminescence on a second time scale and delayed fluorescence on the nanosecond time scale at − 196 °C. Even though there were significant differences between the amino acid sequences of extrinsic proteins in G. violaceus and Synechocystis sp. PCC 6803, the oxygen-evolving activities were similar. The delayed luminescence oscillation patterns and glow curves of thermoluminescence were essentially identical between the two species, and the nanosecond delayed fluorescence spectral profiles and lifetimes were also very similar. These results indicate clearly that even though the oxygen-evolving reactions are carried out in the periplasm by components with altered amino acid sequences, the essential reaction processes for water oxidation are highly conserved. In contrast, we observed significant changes on the reduction side of photosystem II. Based on these data, we discuss the oxygen-evolving activity of G. violaceus.     

Some effects of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785) on the development of chloroplast thylakoid membranes in Hordeum vulgare L.

Chloroplast ultrastructural and photochemical features were examined in 6-d-old barley (Hordeum vulgare L. cv. Sundance) plants which had developed in the presence of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785). In spite of a substantial modification of the fatty-acid composition of thylakoid lipids there were no gross abnormalities in chloroplast morphology, and normal amounts of membrane and chlorophyll were present. Fluorescence kinetics at 77K demonstrated considerable energetic interaction of photosystem (PS)I and PSII chlorophylls within the altered lipid environment. An interference with electron transport was indicated from altered room-temperature fluorescence kinetics at 20°C. Subtle changes in the arrangements of chloroplast membranes were consistently evident and the overall effects of these changes was to increase the proportion of appressed to nonappressed membranes. This correlated with a lower chlorophyll a/b ratio, an increase in the amount of light-harvesting chlorophylls as determined by gel electrophoresis and fluorescence emission spectra, and an increase in excitation-energy transfer from PSII to PSI, as predicted from current ideas on the organisation of photosystems in appressed and non-appressed thylakoid membranes.Abbreviations CP1 P700-chlorophyll a protein - F_o, F_m, F_v minimal, maximal and variable fluorescence yield - LHCP light-harvesting chlorophyll-protein complex - PSI, PSII photosystem I, II - San 9785 4-chloro-5(dimethylamino)-2-phenyl-3(2H)-pyridazinone     

A one microsecond component of chlorophyll luminescence suggesting a primary acceptor of system II of photosynthesis different from Q

The kinetics of the luminescence of chlorophyll a in Chlorella vulgaris were studied in the time range from 0.2 μs to 20 μs after a short saturating flash ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 25}\text{ns}$) under various pretreatment including anaerobiosis, flashes, continuous illumination and various additions. A 1 μs luminescence component probably originating from System II was found of which the relative amplitude was maximum under anaerobic conditions for reaction centers in the state SPQ^? before the flash, about one third for centers in the state S⁺PQ^? or SPQ before the flash, and about one tenth for centers in the state S⁺PQ before the flash. S is the secondary donor complex with zero charge; S⁺ is the secondary donor complex with 1 to 3 positive charges; P, the primary donor, is the photoactive chlorophyll a, P-680, of reaction center 2; Q^? is the reduced acceptor of System II, Q. Under aerobic conditions, where an endogenous quencher presumably was active, the luminescence was reduced by a factor two.The 1 μs decay of the luminescence is probably caused by the disappearance of P⁺ formed in the laser flash according to the reaction ZP⁺ → Z⁺P in which Z is the molecule which donates an electron to P⁺ and which is part of S. After addition of hydroxylamine, the 1 μs luminescence component changed with the incubation time exponentially (τ = 27 s) into a 30 μs component; during the same time, the variable fluorescence yield, measured 9 μs after the laser flash, decreased by a factor 2 with the same time constant. Hereafter in a second much slower phase the fluorescence yield decreased as an exponential function of the incubation time to about the dark value; meanwhile the 30 μs luminescence increased about 50% with the same time constant (τ = 7 min). Heat treatment abolished both luminescence components.The 1 μs luminescence component saturated at about the same energy as the System II fluorescence yield 60 μs after the laser flash and as the slower luminescence components. From the observation that the amplitude is maximum if the laser flash is given when the fluorescence yield is high after prolonged anaerobic conditions (state SQ^?), we conclude that the 1 μs luminescence is probably caused by the reaction

$\text{PWQ}{}^{\mathrm{?}}\text{+hv →}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→}\text{P}{}^{\mathrm{+}}\text{W}{}^{\mathrm{?}}\text{Q}{}^{\mathrm{?}}\text{→}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→ PWQ}{}^{\mathrm{?}}\text{+hv}$

in which W is an acceptor different from Q. The presence of S⁺ reduced the luminescence amplitude to about one third. Two models are discussed, one with W as an intermediate between P and Q and another, which gives the best interpretation, with W on a side path.     

The effect of temperature on the primary reaction of chloroplast Photosystem II Evidence for a temperature-dependent back reaction

The primary reaction of Photosystem II has been studied over the temperature range from −196 to −20 °C. The photooxidation of the reaction-center chlorophyll (P680) was followed by the free-radical electron paramagnetic resonance signal of P680⁺, and the photoreduction of the Photosystem II primary electron acceptor was monitored by the C-550 absorbance change.

At temperatures below −100 °C, the primary reaction of Photosystem II is irreversible. However, at temperatures between −100 and −20 °C a back reaction that is insensitive to 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) occurs between P680⁺ and the reduced acceptor.

The amount of reduced acceptor and P680⁺ present under steady-state illumination at temperatures between −100 and −20 °C is small unless high light intensity is used to overcome the competing back reaction. The amount of reduced acceptor present at low light intensity can be increased by adjusting the oxidation-reduction potential so that P680⁺ is reduced by a secondary electron donor (cytochrome b₅₅₉) before P680⁺ can reoxidize the reduced primary acceptor. The photooxidation of cytochrome b₅₅₉ and the accompanying photoreduction of C-550 are inhibited by DCMU. The inhibition of C-550 photoreduction by DCMU, the dependence of P680 photooxidation and C-550 photoreduction on light intensity, and the effect of the availability of reduced cytochrome b₅₅₉ on C-550 photoreduction are unique to the temperature range where the Photosystem II primary reaction is reversible and are not observed at lower temperatures.     

Interactions of herbicides and azidoquinones at a Photosystem II binding site in the thylakoid membrane

6-Azido-5-decyl-2,3-dimethoxy-p-benzoquinone (6-azido-Q₀C₁₀) was found to replace the native plastoquinone at B (the second stable electron acceptor to Photosystem II (PS II)). The 6-azido-Q₁₀C₁₀ would accept electrons from the primary electron-accepting quinone, Q, thus allowing electron transport through PS II to the plastoquinone pool in thylakoids. The synthetic azidoquinone also competes with the PS II herbicides ioxynil and atrazine for binding. This observation strongly favors the hypothesis that PS II herbicides block electron transport by replacing the native quinone which acts as the second electron carrier on the reducing side of PS II (termed B). Covalent linkage of 6-azido-Q₀C₁₀ to its binding environment by ultraviolet irradiation greatly reduces herbicide-binding affinity but does not lead to a loss in herbicide-binding sites. We take this as evidence that covalent attachment of 6-azido-Q₀C₁₀ allows some freedom of quinone head-group movement such that the herbicides can enter the binding site. This indicates that the protein determinants which regulate quinone and herbicide binding are very closely related, but not identical. A compound somewhat related to 6-azido-Q₀C₁₀ is 2-azido-3-methoxy-5-geranyl-6-methyl-p-benzoquinone (2-azido-Q₂). This compound was found to be an ineffective competitor with respect to herbicide binding. Thus, interactions with protein-binding determinants are highly dependent on the molecular structure of quinones. The 2-azido-Q₂ was an inhibitor of electron flow in the intersystem portion of the chain.     

A study on the lateral distribution of the plastoquinone pool with respect to Photosystem II in stacked and unstacked spinach chloroplasts

The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl₂) and largely unstacked (0.25 mM MgCl₂) membranes. A method to correct for spillover or other quenching changes at the different MgCl₂ concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl₂ concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.     

Studies on the mechanism of ADRY agents (agents accelerating the deactivation reactions of water-splitting enzyme system Y) on thermoluminescence emission

The effect of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT-2p), known to be the most powerful ADRY agent (Renger, G. (1972) Biochim. Biophys. Acta 256, 428–439), on thermoluminescence has been investigated. Two thermoluminescence bands were analyzed: (a) the emission peaking at about 20–30°C caused by warming up of untreated chloroplasts, illuminated with a single 5 μs flash at room temperature and frozen rapidly to 77 K; and (b) the band emitted in the range of ?10 up 10°C after warming of chloroplast suspensions containing 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) which were illuminated with a single 5 μs flash at ?15°C and frozen rapidly at 77 K. These bands were attributed to the recombination of the B ^?S₂(S₃) and X-320 ^?S₂ states, respectively (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). It was found that: (1) The B ^?S₂(S₃) band is markedly diminished at very low ANT-2p concentrations of less than one molecule per 2000 chlorophylls. (2) The inhibition of the X-320 ^?S₂ band requires significantly higher concentrations of ANT-2p (50% peak reduction at one ANT-2p molecule per 100 chlorophylls). (3) Preflashing at room temperature before cooling to ?15°C diminishes the X-320 ^?S₂ band significantly in the presence of ANT-2p, while almost no effect is observed in its absence. (4) The state X-320 ^?S₂ decays monoexponentially with a half-lifetime of 2 min at ?15°C in the absence of ANT-2p. In the presence of one ANT-2p molecule per 800 chlorophylls the decay becomes biphasic with half-lifetimes of 0.5 and 2 min and an amplitude ratio of 2:3, respectively. The results obtained can be explained consistently by the function of ANT-2p as an ADRY agent acting as a mobile species within the thylakoid membrane at room temperature. At subzero temperatures, a ‘fixed-place’ mechanism appears to be operative. The implications for the ADRY effect and thermoluminescence are discussed.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

Role of thylakoid protein kinases in photosynthetic acclimation

Photosynthetic organisms are able to adjust to changes in light quality through state transition, a process which leads to a balancing of the light excitation energy between the antennae systems of photosystem II and photosystem I. A genetic approach has been used in Chlamydomonas with the aim of elucidating the signaling chain involved in state transitions. This has led to the identification of a small family of Ser-Thr protein kinases associated with the thylakoid membrane and conserved in algae and land plants. These kinases appear to be involved both in short and long term adaptations to changes in the light environment.