Picosecond fluorescence decay studies on water-stressed pea leaves: energy transfer and quenching processes in photosystem 2

Detached leaves of pea (Pisum sativum) were submitted to water stress at different relative air humidities. The photosynthetic activity of photosystem 2 (PS2) was monitored by time-resolved picosecond chlorophyll (Chl) fluorescence spectroscopy. In the first days the well-known fast Chl fluorescence decay was observed which indicated high PS2 activity. After a few days the average fluorescence decay time τm reached a maximum, depending on the wilting conditions, but always at a relative loss of leaf mass of 80%. After this maximum, τm decreased within a few hours, the fluorescence decay became similar to that one of an intact leaf, but an additional fluorescence decay component with a lifetime of 3.6 ns appeared. At first the primary quinone QA was reduced due to inhibition of the electron transfer to the secondary quinone QB. Simultaneously, water deficiency caused an electron lack at the oxidizing site of PS2. This disabled the primary electron donor of PS2, tyrosine Z, from reducing the oxidized reaction centre of PS2 (P680+). Thus a recombination of P680+-pheophytin-QA- took place, and the energy was lost as heat. With further water stress, QA was decoupled from PS2. The new fluorescence decay component could therefore be assigned to energetically decoupled antenna complexes.     

Inhibition of photosystem II of spinach by the respiration inhibitors piericidin A and thenoyltrifluoroacetone

The effects of several respiration inhibitors on photosystem II (PS II) were investigated. Among the agents tested, piericidin A and thenoyltrifluoroacetone (TTFA) inhibited the photosynthetic electron transport of spinach as measured from chlorophyll (Chl) fluorescence parameters (Fm'-F)/Fm' and Fv/Fm. Using specific donors and acceptors of electrons, we identified the sites of inhibition in and around the PS II complex; the site of inhibition by TTFA was between QA, primary quinone acceptor in PS II, and QB, secondary quinone acceptor, in the acceptor side of P680, the reaction center Chl of PS II, while inhibition by piericidin A of the acceptor side was downstream of Q(B), out of the PS II complex. Both agents also inhibited the donor side of P680, probably between tyrosine-161 of the reaction center protein of PS II and P680.     

Investigations on the electron transfer processes in Photosystem II: New insights by chlorophyll fluorescence decay measurements with additional saturating light pulses

Electron transfer processes in leaves were investigated by chlorophyll fluorescence decay measurements. A fast chlorophyll fluorescence decay was observed in the intact state, reflecting normal electron transfer in Photosystem II. After treatment with DCMU a slow chlorophyll fluorescence decay was measured due to blocked electron transfer after the primary quinone Q_A. Additional saturating light pulses, one between each two measuring pulses, were used to completely reduce Q_A of the intact leaf: the chlorophyll fluorescence decay became similar to that of a DCMU treated leaf. A decreased electron donation rate to the reaction centre P680 was obtained after treatment with hydroxylamine. The intensity of the additional saturating light pulses was not sufficient to reduce all Q_A under this condition and only a small increase of the average chlorophyll fluorescence decay time occurred. Following our previous paper [Berg et al. (1997) Photosynthetica 34, in press], we investigated the effects of water stress with the additional saturating light pulses. An almost complete reduction of Q_A was possible after water stress started. A small, but systematic shortening of the slow chlorophyll fluorescence decay followed, up to a relative loss of leaf mass of 80%. At this time a rapid shortening of the chlorophyll fluorescence decay occurred, caused by an electron deficiency at the donor site of PS II. Additional saturating light pulses had no effects on the chlorophyll fluorescence decay any more, revealing a radiationless recombination between the reduced primary quinone Q and the oxidized reaction centre P680⁺.     

Chlorophyll a fluorescence transient as an indicator of active and inactive Photosystem II in thylakoid membranes

Upon illumination, a dark-adapted photosynthetic sample shows time-dependent changes in chlorophyll (Chl) a fluorescence yield, known as the Kautsky phenomenon or the OIDPS transient. Based on the differential effects of electron acceptors such as 2,5-dimethyl-p-benzoquinone (DMQ) and 2,6-dichloro-p-benzoquinone (DCBQ) on Chl a fluorescence transients of spinach thylakoids, we suggest that the OID phase reflects the reduction of the electron acceptor QA to QA- in the inactive PS II (see Graan, T. and Ort, D. (1986) Diochim. Biophys. Acta 852, 320-330). In spinach thylakoids, heat-induced increase of the Chl a fluorescence yield is also differentially sensitive to the addition of DMQ and DCBQ suggesting that this increase is mainly on the 'I' level, and thus heating is suggested to convert active PS II to inactive PS II centers. The kinetics of decay of QA-, calculated from variable Chl a fluorescence, was analyzed into three exponential components (365-395 microseconds; 6-7 ms; and 1.4-1.7 s). In heated samples, the decay rate of variable Chl a fluorescence is slower than the normal back-reaction rate; there is a preponderance of the slow component that may be due, partly, to the active centers undergoing slow back reaction between QA- and the S2 state of the oxygen-evolving complex.     

Inhibition of photosynthetic oxygen evolution and electron transfer from the quinone acceptor Q<Subscript>A</Subscript><Superscript>−</Superscript> to Q<Subscript>B</Subscript> by iron deficiency

The effect of iron deficiency on photosynthetic electron transport in Photosystem II (PS II) was studied in leaves and thylakoid membranes of lettuce (Lactuca sativa, Romaine variety) plants. PS II electron transport was characterized by oxygen evolution and chlorophyll fluorescence parameters. Iron deficiency in the culture medium was shown to affect water oxidation and the advancement of the S-states. A decrease of maximal quantum yield of PS II and an increase of fluorescence intensity at step J and I of OJIP kinetics were also observed. Thermoluminescence measurements revealed that charge recombination between the quinone acceptor of PS II, Q_B, and the S₂ state of the Mn-cluster was strongly perturbed. Also the dark decay of Chl fluorescence after a single turnover white flash was greatly retarded indicating a slower rate of Q_A ⁻ reoxidation.     

Energetics of primary and secondary electron transfer in Photosystem II membrane particles of spinach revisited on basis of recombination-fluorescence measurements

Photon absorption by one of the roughly 200 chlorophylls of the plant Photosystem II (PSII) results in formation of an equilibrated excited state (Chl200*) and is followed by chlorophyll oxidation (formation of P680+) coupled to reduction of a specific pheophytin (Phe), then electron transfer from Phe- to a firmly bound quinone (QA), and subsequently reduction of P680+ by a redox-active tyrosine residue denoted as Z. The involved free-energy differences (DeltaG) and redox potentials are of prime interest. Oxygen-evolving PSII membrane particles of spinach were studied at 5 degrees C. By analyzing the delayed and prompt Chl fluorescence, we determined the equilibrium constant and thus free-energy difference between Chl200* and the [Z+,QA-] radical pair to be -0.43+/-0.025 eV, at 10 mus after the photon absorption event for PSII in its S(3)-state. On basis of this value and previously published results, the free-energy difference between P680* and [P680+,QA-] is calculated to be -0.50+/-0.04 eV; the free-energy loss associated with electron transfer from Phe to QA is found to be 0.34+/-0.04 eV. The given uncertainty ranges do not represent a standard deviation or likely error, but an estimate of the maximal error. Assuming a QA-/QA redox potential of -0.08 V, the following redox-potential estimates are obtained: +1.25 V for P680/P680+; +1.21 V for Z/Z+ (at 10 mus); -0.42 V for Phe-/Phe; -0.58 V for P680*/P680+.     

水葫芦及菠菜光合作用原初光反应的超快光谱研究

采用相同的分离技术,从水葫芦(Eichhornia crassipes(Mart)Solms.)和菠菜(Spinacia oleracea L.)叶片中提取叶绿体.利用吸收光谱和低温荧光光谱及皮秒荧光单光子计数技术对它们的光谱性质和光系统Ⅱ荧光寿命进行了研究.这两种叶绿体吸收光谱相似,暗示着它们都能高效吸收不同波长的光子.低温荧光光谱显示,水葫芦叶绿体两个光系统之间激发能分配平衡状态差,表明不利于该植物叶绿体高效利用吸收的光子能.采用三指数动力学模型对测定的光系统Ⅱ荧光衰减曲线拟合,水葫芦叶绿体光系统Ⅱ荧光衰减寿命分别是:138,521和1 494 ps;菠菜叶绿体荧光寿命分别是:197,465和1 459ps.并且归属了荧光组分,慢速度荧光衰减是由叶绿素堆积造成的,中等速度荧光衰减源于PSⅡ反应中心重新结合电荷组分,快速度荧光衰减归属于PSⅡ反应中心组分.基于20ps模型计算的水葫芦和菠菜叶绿体PSⅡ反应中心激发能转能效率分别是87%和91%.该结果与转能效率为100%的观点不一致.实验结果支持PSⅡ反应中心电荷分裂20 ps时间常数模型.根据转能效率,水葫芦生长速度不大于菠菜生长速度,但是,水葫芦叶绿体中含有丰富的胡萝卜素成分,其单位质量叶绿体吸收光能大于单位质量菠菜叶绿体吸收的量.实验结果还暗示植物叶绿体体系传能高效,接近于100%.     

Multiple redox-active chlorophylls in the secondary electron-transfer pathways of oxygen-evolving photosystem II

Photosystem II (PS II) is unique among photosynthetic reaction centers in having secondary electron donors that compete with the primary electron donors for reduction of P680(+). We have characterized the photooxidation and dark decay of the redox-active accessory chlorophylls (Chl) and beta-carotenes (Car) in oxygen-evolving PS II core complexes by near-IR absorbance and EPR spectroscopies at cryogenic temperatures. In contrast to previous results for Mn-depleted PS II, multiple near-IR absorption bands are resolved in the light-minus-dark difference spectra of oxygen-evolving PS II core complexes including two fast-decaying bands at 793 and 814 nm and three slow-decaying bands at 810, 825, and 840 nm. We assign these bands to chlorophyll cation radicals (Chl(+)). The fast-decaying bands observed after illumination at 20 K could be generated again by reilluminating the sample. Quantization by EPR gives a yield of 0.85 radicals per PS II, and the yield of oxidized cytochrome b 559 by optical difference spectroscopy is 0.15 per PS II. Potential locations of Chl(+) and Car(+) species, and the pathways of secondary electron transfer based on the rates of their formation and decay, are discussed. This is the first evidence that Chls in the light-harvesting proteins CP43 and CP47 are oxidized by P680(+) and may have a role in Chl fluorescence quenching. We also suggest that a possible role for negatively charged lipids (phosphatidyldiacylglycerol and sulfoquinovosyldiacylglycerol identified in the PS II structure) could be to decrease the redox potential of specific Chl and Car cofactors. These results provide new insight into the alternate electron-donation pathways to P680(+).     

Enhanced photosystem 2 thermostability during leaf growth of Elm (Ulmus pumila) seedlings

We examined photosynthetic activities and thermostability of photosystem 2 (PS2) in leaves of elm (Ulmus pumila) seedlings from initiation to full expansion. During leaf development, net photosynthetic rate (P _N) increased gradually and reached the maximum when leaves were fully developed. In parallel with the increase of P _N, chlorophyll (Chl) content was significantly elevated. Chl a fluorescence measurements showed that the maximum quantum yield of PS2 (ϕ_PS2), the efficiency a trapped exciton, moved an electron into the electron transport chain further than Q_A ⁻ (Ψ_o), and the quantum yield of electron transport beyond Q_A (ϕ_Eo) increased gradually. These results were independently confirmed by our low irradiance experiments. When subjected to progressive heat stress, the young leaves exhibited considerably lower ϕ_PS2 and higher minimal fluorescence (F₀) than the mature leaves, revealing the highly sensitive nature of PS2 under heat in the newly initiating leaves. Further analysis showed that PS2 structure in the newly initiating leaves was strongly altered under heat, as evidenced by the increased fluorescence signals at the position of the K step. We therefore demonstrated an inhibition in the oxygen-evolving complex (OEC) in the young leaves. This resulted in decrease in amount of the functional PS2 reaction centres and relative increase in the PS2 reaction centres with inhibited electron transport at the acceptor side under heat. We suggest that the enhanced thermostability of PS2 during leaf development is associated with improved OEC stability.     

Tetranitromethane modification of photosystem 2

Inhibition of photosystem 2 by the peptide-modification reagent, tetranitromethane, has been investigated with spinach digitonin particles. In the presence of tetranitromethane, (1) the initial fluoresence yield is suppressed with a concomitant elimination of the variable component of fluorescence; (2) the optical absorption transient at 820 nm, attributed to P680⁺, is greatly attenuated; (3) diphenylcarbazide-supported photoreduction of dichlorophenol indophenol is abolished; and (4) electron spin resonance Signal 2f and Signal 2s are eliminated. These results are consistent with multiple sites of modification in photosystem 2 by tetranitromethane, and suggest further that this reagent can inhibit charge stabilization in the reaction center.Abbreviations D₁ electron donor to P680⁺ in oxygen-inhibited photosystem 2 preparations - DPIP 2,6-dichlorophenol indophenol - esr electron spin resonance - F_i initial chlorophyll a fluorescence yield - F_max maximum chlorophyll a fluorescence yield - F_v variable chlorophyll a fluorescence yield - FWHM full width at half maximum - Mes 2-(N-morpholino)ethanesulfonic acid - P680 primary electron donor chlorophyll of photosystem 2 - Ph pheophytin - PS 2-photosystem 2 - Q_a primary quinone electron acceptor - Q_b secondary quinone acceptor - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine - TNM tetranitromethane     

Energetics of primary and secondary electron transfer in Photosystem II membrane particles of spinach revisited on basis of recombination-fluorescence measurements

Photon absorption by one of the roughly 200 chlorophylls of the plant Photosystem II (PSII) results in formation of an equilibrated excited state (Chl200*) and is followed by chlorophyll oxidation (formation of P680+) coupled to reduction of a specific pheophytin (Phe), then electron transfer from Phe− to a firmly bound quinone (QA), and subsequently reduction of P680+ by a redox-active tyrosine residue denoted as Z. The involved free-energy differences (ΔG) and redox potentials are of prime interest. Oxygen-evolving PSII membrane particles of spinach were studied at 5 °C. By analyzing the delayed and prompt Chl fluorescence, we determined the equilibrium constant and thus free-energy difference between Chl200* and the [Z+,QA−] radical pair to be −0.43 ± 0.025 eV, at 10 μs after the photon absorption event for PSII in its S₃-state. On basis of this value and previously published results, the free-energy difference between P680* and [P680+,QA−] is calculated to be −0.50 ± 0.04 eV; the free-energy loss associated with electron transfer from Phe to QA is found to be 0.34 ± 0.04 eV. The given uncertainty ranges do not represent a standard deviation or likely error, but an estimate of the maximal error. Assuming a QA−/QA redox potential of −0.08 V [Krieger et al., 1995, Biochim. Biophys. Acta 1229, 193], the following redox-potential estimates are obtained: +1.25 V for P680/P680+; +1.21 V for Z/Z+ (at 10 μs); −0.42 V for Phe−/Phe; −0.58 V for P680*/P680+.     

The quenching characteristics of potassium iridic chloride and their meaning for the origin of chlorophyll fluorescence components

To understand the origins of the different lifetime components of photosystem 2 (PS2) chlorophyll (Chl) fluorescence we have studied their susceptibility to potassium iridic chloride (K₂IrCl₆) which has been shown to bleach antenna pigments of photosynthetic bacteria (Loach et al. 1963). The addition of K₂IrCl₆ to PS2 particles gives rise to a preferential quenching of the variable Chl fluorescence (F_v). At concentrations lower than 20 M, this is brought about mainly by a decrease in the yield, but not in the lifetime, of the slowest component when all the PS2 reaction centres are closed (F_M). The yield of the middle and fast decays are not significantly altered. This type of quenching is not seen with DNB. The iridate-induced quenching of the initial fluorescence level (F₀) is due to a proportional decrease in the yield and lifetime of the three components and correlates with the observed modification in the relative quantum yield of oxygen evolution. In this concentration range a bleaching of Chl a is seen. At higher iridate levels, greater than 20 M, a proportional decrease in the lifetimes and yields of the three kinetic components is seen at F_M. These changes are associated with a carotenoid bleaching. In isolated light harvesting Chl a/b complexes of PS2 (LHC2), iridate addition converts a 4 ns decay into a 200 ps emission and both types of bleaching are observed. By also measuring the rate of PS2 trap closure versus iridate concentration, we have discussed the results in terms of excitation energy transfer.Abbreviations DNB m-dinitrobenzene - F_M maximum Chl fluorescence - F₀ initial fluorescence - F_v variable fluorescence - I pheophytin a primary electron acceptor of PS2 - P₆₈₀ chlorophyll a of photochemical centre - PS2 photosystem 2 - Q_A primary stable electron acceptor of PS2 - Chl chlorophyll - LHC2 light harvesting Chl a/b complex of PS2 - MES 2(N-morpholino) ethanesulfonic acid - DCMU 3-(3-4-dichlorophenyl) 1-1 dimethylurea - PPBQ phenyl-p-benzo-quinone - BBY PS2-enriched membranes prepared as in Berthold et al. (1981) - Q₄₀₀ PS2 electron acceptor with a midpoint potential of 400 mV     

Decrease of Fluorescence Intensity After the K Step in Chlorophyll a Fluorescence Induction is Suppressed by Electron Acceptors and Donors to Photosystem 2

Chlorophyll a fluorescence induction measured by a fluorometer with a high temperature stressed plant material shows a new K step which is a clear peak due to fast fluorescence rise and subsequent decrease of fluorescence intensity. We focused on an explanation of the decrease of fluorescence after the K step using artificial electron acceptors and donors to photosystem 2 (PS2). Addition of the artificial electron acceptors or donors suppressed the decrease of fluorescence after the K step. We suggest that the decrease mainly reflects (by more than 81 %) an energy loss process in the reaction centre of PS2 which is most probably a nonradiative charge recombination between P680⁺ (oxidised primary electron donor in PS2) and a negative charge stored on either Pheo⁻ or Q_A ⁻ (reduced primary electron acceptor of PS2 and reduced primary quinone electron acceptor of PS2, respectively). We suggest that the energy loss process is only possible when the inhibition of both the donor and the acceptor sides of PS2 occurs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Light-induced quenching of chlorophyll fluorescence at 77 K in leaves,chloroplasts and Photosystem II particles

The light-induced chlorophyll (Chl) fluorescence decline at 77 K was investigated in segments of leaves, isolated thylakoids or Photosystem (PS) II particles. The intensity of chlorophyll fluorescence declines by about 40% upon 16 min of irradiation with 1000 μmol m⁻² s⁻¹ of white light. The decline follows biphasic kinetics, which can be fitted by two exponentials with amplitudes of approximately 20 and 22% and decay times of 0.42 and 4.6 min, respectively. The decline is stable at 77 K, however, it is reversed by warming of samples up to 270 K. This proves that the decline is caused by quenching of fluorescence and not by pigment photodegradation. The quantum yield for the induction of the fluorescence decline is by four to five orders lower than the quantum yield of Q_A reduction. Fluorescence quenching is only slightly affected by addition of ferricyanide or dithionite which are known to prevent or stimulate the light-induced accumulation of reduced pheophytin (Pheo). The normalised spectrum of the fluorescence quenching has two maxima at 685 and 695 nm for PS II emission and a plateau for PS I emission showing that the major quenching occurs within PS II. ‘Light-minus-dark’ difference absorbance spectra in the blue spectral region show an electrochromic shift for all samples. No absorbance change indicating Chl oxidation or Pheo reduction is observed in the blue (410–600 nm) and near infrared (730–900 nm) spectral regions. Absorbance change in the red spectral region shows a broad-band decrease at approximately 680 nm for thylakoids or two narrow bands at 677 and 670–672 nm for PS II particles, likely resulting also from electrochromism. These absorbance changes follow the slow component of the fluorescence decline. No absorbance changes corresponding to the fast component are found between 410 and 900 nm. This proves that the two components of the fluorescence decline reflect the formation of two different quenchers. The slow component of the light-induced fluorescence decline at 77 K is related to charge accumulation on a non-pigment molecule of the PS II complex. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of photosystems 2 and 1 during leaf growth in grapevine seedlings probed by chlorophyll a fluorescence transient and 820 nm transmission in vivo

Chlorophyll (Chl) a fluorescence transient and 820-nm transmission kinetic were investigated to explore the development of photosynthetic apparatus in grapevine leaves from emergence to full expansion. In this study, all leaves at various developing stages exhibited typical Chl a fluorescence transient. In newly initiating leaves, the maximum quantum yield of primary photochemistry (ϕ_P0) was slightly lower (<10 %) than that in fully expanded leaves. Nevertheless, the fluorescence rise from O to J step was clearly speeded up in young leaves compared with that in fully expanded leaves. Additionally, a distinct K step appeared in young leaves at high irradiances. With leaf development, the efficiency that a trapped exciton can move an electron into the electron transport chain further than Q_A ⁻ (Ψ₀), the quantum yield of electron transport beyond Q_A (ϕ_E0), electron transport flux per excited cross section (ET₀/CS₀), the amount of active photosystem (PS) 2 reaction centres per excited cross section (RC/CS₀), and the performance index on cross section basis (PI_CS) increased gradually and rapidly. Young leaves had strikingly lower amplitude of transmission at 820 nm. A linear relationship between Ψ₀ and the transmission at 820 nm (I₃₀/I₀) was evident. Based on these data, we suggest that (1) the primary photochemistry of PS2 may be not the limiting step of the photosynthetic capacity during leaf growth under natural irradiance; (2) oxygen evolving complex (OEC) might be not fully connected to PS2 at the beginning of leaf growth; (3) though there are a few functional PS1 and PS2 at the early stages of leaf development, they match perfectly.     

Period four oscillations in chlorophyll a fluorescence

The discovery of period four oscillations of the fluorescence yield under flashing light demonstrated that not only the redox state of the Photosystem II (PS II) electron acceptor Q_A, but also the oxygen evolving cycle (described by the S states) modulates the fluorescence yield of chlorophyll (Chl). The positive charges accumulated on the donor side of PS II act on the fluorescence yield (measured in the Q_A ⁻ state during a strong flash) through the concentration of the quencher P₆₈₀ ⁺, the oxidized form of PS II reaction center Chl a. However, the period four oscillations of the fluorescence yield detected 1 s after a strong flash (in the P₆₈₀Q_A state) have not yet been fully explained. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosynthetic activity of poikilochlorophyllous desiccation tolerant plant <Emphasis Type="Italic">Reaumuria soongorica</Emphasis> during dehydration and re-hydration

Diurnal patterns of gas exchange and chlorophyll (Chl) fluorescence parameters of photosystem 2 (PS2) as well as Chl content were analyzed in Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub during dehydration and rehydration. The net photosynthetic rate (P _N), maximum photochemical efficiency of PS2 (variable to maximum fluorescence ratio, F_v/F_m), quantum efficiency of non-cyclic electron transport of PS2, and Chl content decreased, but non-photochemical quenching of fluorescence and carotenoid content increased in stems with the increasing of drought stress. 6 d after re-hydration, new leaves budded from stems. In the re-watered plants, the chloroplast function was restored and Chl a fluorescence returned to a similar level as in the control plants. This improved hydraulic adjustment in plant triggered a positive effect on ion flow in the tissues and increased shoot electrical admittance. Thus R. soongorica plants are able to sustain drought stress through leaf abscission and keep part of Chl content in stems.     

Interaction of linolenic acid with bound quinone molecules in Photosystem II. Time-resolved optical and electron spin resonance studies

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance spectroscopy, have been utilized to monitor electron-transport activity in Photosystem II subchloroplast particles. These studies have indicated that in the presence of 100 microM linolenic acid (1) a high initial fluorescence yield (Fi) is observed upon steady-state illumination of the dark-adapted sample; (2) flash-induced absorption transients (t greater than 10 mus) in the region of 820 nm, attributed to P-680+, are first slowed, then abolished; and (3) electron spin resonance Signal IIs and Signal IIf (Z+) are not detectable. Upon reversal of linolenic acid inhibition by washing with bovine serum albumin, optical and electron spin resonance transients originating from the photooxidation of P-680 are restored. Similarly, the variable component of fluorescence is recovered with an accompanying restoration of Signal IIs and Signal IIf. The data indicate that linolenic acid affects two inhibition sites in Photosystem II: one located between pheophytin and QA on the reducing side, and the other between electron donor Z and P-680 on the oxidizing side. Since both sites are associated with bound quinone molecules, we suggest that linolenic acid interacts at the level of quinone binding proteins in Photosystem II.     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Photoinhibition of hydroxylamine-extracted photosystem II membranes: identification of the sites of photodamage.

Electron paramagnetic resonance (EPR) analyses (g = 2 region) and optical spectrophotometric analyses of P680+ were made of NH2OH-extracted photosystem II (PSII) membranes after various durations of weak-light photoinhibition, in order to identify the sites of damage responsible for the observed kinetic components of the loss of electron transport [Blubaugh, D.J., & Cheniae, G.M. (1990) Biochemistry 29, 5109-5118]. The EPR spectra, recorded in the presence of K3Fe(CN)6, gave evidence for rapid (t1/2 = 2-3 min) and slow (t1/2 = 3-4) losses of formation of the tyrosyl radicals YZ+ and YD+, respectively, and the rapid appearance (t1/2 = 0.8 min) of a 12-G-wide signal, centered at g = 2.004, which persisted at 4 degrees C in subsequent darkness in rather constant abundance (approximately 1/2 spin per PSII). This latter EPR signal is correlated with quenching of the variable chlorophyll a fluorescence yield and is tentatively attributed to a carotenoid (Car) cation. Exogenous reductants (NH2OH greater than or equal to NH2NH2 greater than DPC much greater than Mn2+) were observed to reduce the quencher, but did not reverse other photoinhibition effects. An additional 10-G-wide signal, tentatively attributed to a chlorophyll (Chl) cation, is observed during illumination of photoinhibited membranes and rapidly decays following illumination. The amplitude of formation of the oxidized primary electron donor, P680+, was unaffected throughout 120 min of photoinhibition, indicating no impairment of charge separation from P680, via pheophytin (Pheo), to the first stable electron acceptor, QA. However, a 4-microsecond decay of P680+, reflecting YZ----P680+, was rapidly (t1/2 = 0.8 min) replaced by an 80-140 microsecond decay, presumably reflecting QA-/P680+ back-reaction. Photoinhibition caused no discernible decoupling of the antenna chlorophyll from the reaction center complex. We conclude that the order of susceptibility of PSII components to photodamage when O2 evolution is impaired is Chl/Car greater than YZ greater than YD much greater than P680, Pheo, QA.