Donor function of phosphate ions in photosystem 2

Evidence was obtained for the interaction between the photosystem 2 (PS2) reaction centre (RC) chlorophyll (Chl) P680 and inorganic phosphate, Pi. The light-induced endogenous basal electron transport to ferricyanide in PS2 depended on endogenous Pi. The electron transport in phosphate deficient chloroplasts was absent, and could be resumed upon the addition of exogenous Pi or of the exogenous electron donor, diphenylcarbazide. Some chloroplast Chl molecules were apparently bound with Pi to a complex via the magnesium atom that was detected by the increase in absorbance in the Chl a absorption maximum at 435 nm observed after the consumption of endogenous Pi in the photophosphorylation reactions. The electron paramagnetic resonance (EPR) Signal I, found in the spectra at 77 K after irradiation of frozen samples in chloroplasts poor in endogenous Pi, was the sum of P700+ and P680+ signals. The P680+ signal disappeared after addition of Pi, diphenylcarbazide or diuron to the chloroplasts before freezing. In addition, the EPR doublet signal of the phosphate anion radicals was recorded at 77 K after irradiation in the ethanol solutions of Chl a containing potassium phosphate. The same doublet signal was discovered in the difference EPR spectrum "chloroplasts minus chloroplasts with diuron" at 77 K after irradation. The results are a possible evidence of the participation of phosphate ions in the primary light reactions of PS2.     

Participation of inorganic phosphates as electron donors in the primary reactions of photosynthesis in Rhodobacter sphaeroides

The formation of ATP during photophosphorylation in chromatophores from purple nonsulfur bacterium Rhodobacter sphaeroides in the presence of phenazine methosulfate and without exogenous electron carriers under constant illumination and by the action of single light flashes was studied. It was shown that the photoinduced transport of electrons to the exogenous electron acceptor depends on phosphate. It was assumed that phosphate ions are electron donors in the reaction center P870; by the action of light, P870 converts the phosphate ion HPO4(2-) into anion radical HPO4-.. In the difference EPR spectra "light minus darkness" at 77 K, an asymmetrical doublet signal with a weak low-field line was observed. The signal had a g-tensor of about 2.014 and a hyperfine coupling constant of about 2.5 mT and belongs probably to the phosphate anion radical.     

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(+).     

Photoinactivation of the reactivation capacity of photosystem II in pea subchloroplast particles after a complete removal of manganese

After a complete removal of Mn from pea subchloroplast photosystem-II (PS II) preparations the electron phototransfer and oxygen evolution are restored upon addition of Mn²⁺ and Ca²⁺. Pre-illumination of the sample in the absence of Mn²⁺ leads to photoinhibition (PI) — irreversible loss of the capability of PS II to be reactivated by Mn²⁺. The effect of PI is considerably decreased in the presence of Mn²⁺ (4 Mn atoms per reaction center of PS II) and it is increased in the presence of ferricyanide or p-benzoquinone revealing the oxidative nature of the photoeffect. PI results in suppression of oxygen evolution, variable fluorescence, photoreduction of 2,6-dichlorophenol indophenol from either water or diphenylcarbazide. However, photooxidation of chlorophyll P₆₈₀, the primary electron donor of PS II as well as dark and photoinduced EPR signal II (ascribed to secondary electron donors D ₁ and Z) are preserved. PI is accompanied by photooxidation of 2–3 carotenoid molecules per PS II reaction center (RC) that is accelerated in the presence of ferricyanide and is inhibited upon addition of Mn²⁺ or diuron. The conclusion is made that PI in the absence of Mn leads to irreversible oxidative inactivation of electron transfer from water to RC of PS II which remains photochemically active. A loss of functional interaction of RC with the electron transport chain as a common feature for different types of PS II photoinhibition is discussed.Abbreviations A photoinduced absorbance changes - DPC diphenylcarbazide - DPIP 2,6-dichlorophenol indophenol - F _o constant fluorescence of chlorophyll - F photoinduced changes of Chl fluorescence yield - Mn manganese - P₆₈₀ the primary electron donor in PS II - PI photoinhibition - PS II photosystem II - Q the primary (quinone) electron acceptor in PS II - RC reaction center     

Inhibition of the phosphate transporter during isolation of intact chloroplasts from leaves of sunflower

Chloroplasts isolated from spinach leaves by the mechanical method were intact and exhibited high rates of CO₂-dependent oxygen evolution whereas chloroplasts isolated from sunflower leaves by the same technique were also intact but showed only low rates of oxygen evolution. The rate of uptake of orthophosphate (Pi) from the suspending medium with sunflower chloroplasts was less than 20% of that in spinach chloroplasts. The apparent Km for Pi transport was lower in sunflower chloroplasts but uptake was competitively inhibited by 3-phosphoglycerate in chloroplasts from both species. Uptake of malate (via the dicarboxylate transporter) and of ATP (via the adenine nucleotide transporter) was also reduced in sunflower chloroplasts compared to spinach chloroplasts. The endogenous Pi content and total exchangeable phosphate pool of sunflower chloroplasts were less than half that in spinach chloroplasts.Addition of a number of possible protective agents to the grinding medium failed to prevent the loss of photosynthetic activity during mechanical isolation of sunflower chloroplasts. Grinding mixtures of spinach and sunflower leaves together indicated that spinach chloroplasts were not inhibited by the sunflower leaf extract. Chloroplasts isolated from sunflower leaves via protoplasts had high rates of CO₂-dependent oxygen evolution. The Vmax and Km for Pi uptake, endogenous Pi content and total exchangeable phosphate pool of chloroplasts isolated from sunflower protoplasts were all similar to spinach chloroplasts. It is concluded that inner envelope membrane proteins are damaged during mechanical isolation of sunflower chloroplasts. The decrease in activity of the phosphate transporter and loss of endogenous phosphate may contribute to the low rates of photosynthesis observed in chloroplasts isolated by the mechanical method from leaves of sunflower and possibly other species.Abbreviations PGA 3-phosphoglyceric acid     

Electron transfer in photosystem II at cryogenic temperatures

The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic characterization of triplet forming states in photosystem II.

Fluorescence and electron paramagnetic resonance (EPR) measurements have been applied to characterize chlorophyll triplet formation in the reaction center of photosystem II (PSII). A highly triplet forming state was generated in PSII membranes by chemical double reduction of the primary electron acceptor QA. In triplet forming PSII centers, the steady-state yield of chlorophyll fluorescence decreased to about 70% of the maximal fluorescence yield observed in closed PSII centers in which QA is singly reduced. The results are well interpreted in the framework of a model where the charge state of QA electrostatically controls the yield of primary charge separation [Schatz, G. H., Brock, H., & Holzwarth, A. R. (1988) Biophys. J. 54, 397-405]. Thus, high triplet yield and decreased, although still quite high, fluorescence indicate a charge-neutralized state of PSII in which QA is singly or doubly reduced and protonated or absent. The EPR signal of the triplet primary chlorophyll donor, 3P680, is suppressed by illumination at 77 K concomitant with the formation of a cationic radical (g = 2.0025-2.0027, and 0.92 mT wide) that is stable in the dark. This is attributed to the oxidation of an accessory chlorophyll (Chl) in the vicinity of P680. Electrostatic repulsion between Chl+ and P680+ is likely to prevent primary charge separation, and in turn triplet formation, providing a further example of electrostatic control of primary charge separation. The triplet P680 EPR signal is also suppressed in the presence of oxygen. This effect, which is almost completely reversible by removing the oxygen, is attributed to the interaction of triplet P680 with triplet O2.     

Hyperfine structure of the photoexcited triplet state 3P680 in plant PS II reaction centres as determined by pulse ENDOR spectroscopy

The triplet states in plant photosystem II (PS II), 3P680, and from chlorophyll a, 3Chl a, in organic solution have been investigated using pulse ENDOR combined with repetitive laser excitation at cryogenic temperature with the aim to obtain their hyperfine (hf) structure. The large zero field splitting (ZFS) tensor of 3P680 enabled orientation selection via the electron spin resonance (EPR) field setting along the ZFS tensor axes. ENDOR spectra have been obtained for the first time also for the in-plane X- and Y-orientations of the ZFS tensor. This allowed a full determination of the hf-tensors of the three methine protons and one methyl group of 3P680. Based on the orientations of the axes of these hf-tensors, a unique orientation of the axes of the ZFS tensor of 3P680 in the Chl a molecular frame was obtained. These data serve as a structural basis for determining the orientation of 3P680 in the PS II protein complex by EPR on single crystals (see M. Kammel et al. in this issue). The data obtained represent the first complete set of the larger hf-tensors of the triplet state 3P680. They reflect the spin density distribution both in the highest occupied (HOMO) and lowest unoccupied (LUMO) orbitals. The data clearly confirm that 3P680 is a monomeric Chl a species at low temperature (T=10 K) used, as has been proposed earlier based on D- and E-values obtained from EPR and optically detected magnetic resonance (ODMR) studies. Comparison with the hf data for the cation and anion radicals of Chl a indicates a redistribution of spin densities in particular for the LUMO orbital of the triplet states. The electron spin distribution in the LUMO orbital is of special interest since it harbours the excited electron in the excited P680 singlet state, from which light-induced electron transfer proceeds. Observed shifts of hf couplings from individual nuclei of 3P680 as compared with 3Chl a in organic solution are of special interest, since they indicate specific protein interactions, e.g. hydrogen bonding, which might be used in future studies for assigning 3P680 to a particular chlorophyll molecule in PS II.     

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis     

Photoinhibition affects the non-heme iron center in photosystem II.

Effects on the PS II acceptor side caused by exposure to strong white light (180 W/m2) of PS II membrane fragments (spinach) at pH 6.5 and 0 degrees C were analyzed by measuring low temperature EPR signals and flash-induced transient changes of the fluorescence quantum yield. The following results were obtained: (a) the extent of the light induced g = 1.9 EPR signal as a measure of photochemical Fe2+QA- formation declines with progressing photoinhibition. The half-life of this effect is independent of the absence or presence of an exogenous electron acceptor during the photoinhibitory treatment; (b) in samples photoinhibited in the absence of an electron acceptor and subsequently incubated with K3[Fe(CN)6] in the dark, the extent of the g = 8 EPR signal (reflecting the oxidized Fe3+ form of the endogenous non-heme iron center) and of the flash-induced change of the fluorescence yield (as a measure of fast electron transfer from QA- to Fe3+ after the first flash; [see (1992) Photosynth. Res. 31, 113-126] exhibits the same dependence on photoinhibition time as the g = 1.9 EPR signal; (c) in samples photoinhibited in the presence of an exogenous electron acceptor, the signals reflecting Fe(3+)-formation and fast electron transfer from QA- to Fe3+ decline faster than the g = 1.9 EPR signal. These results provide for the first time direct evidence that the endogenous non-heme iron center located between QA and QB is susceptible to modifications by light stress. The implications of this finding will be discussed.     

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.     

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein.The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the D1 polypeptide, as shown with PS II membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.Abbreviations atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine - Chl chlorophyll, diuron - (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DPC diphenylcarbazide - ioxynil 4-cyano-2,6-diiodophenol - k_b binding constant - Mes 4-morpholinoethanesulfonic acid - P-680 reaction-center chlorophyll a of photosystem-II - PAGE polyacrylamide gel electrophoresis - PS II photosystem-II - Q_A and Q_B primary and secondary quinone electron acceptors - Z electron donor to the photosystem-II reaction center - SDS sodium dodecylsulfate - Tricine N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine     

华山松吸收光谱及叶绿素荧光特性的地理变异

利用分离的叶绿体作实验材料,发现华山松(Pinus armandi Franch.)南方种源的4阶导数吸收光谱在680nm处峰值较大,在670nm处峰值较小,而北方种源中出现了在680nm处峰值较在670nm峰值小的类群,推断北方种群反应中心活力有下降趋势。南、北种源之间的低温(77K)荧光发射光谱有明显差异,PSⅠ、PSⅡ发射峰位置出现地理变动。低温荧光激发光谱分析表明,地理变异主要发生在叶绿素a的分子状态上。研究还表明,完整的针叶因为有角质层、松脂等物质干扰,检测不出光谱的差异,不是理想的实验材料。     

Geographical Variation in the Characteristics of Absorption Spectra and Fluorescence Spectra of Pinus armandi

By measuring the derivative absorption spectra of chloroplasts of different populations of Pinus armandi Franch., it was found that southern populations maintained higher absorption at 680 nm than at 670 nm, but some of the northern populations deviated the maximum absorption from 680 nm to 670 nm, which indicated that the activity of PS Ⅱ in some of the northern populations declined. Clear geographical differences also have been found in the positions of emission peaks of PS Ⅰ and PS Ⅱ in the fluorescence emission spectra at 77 K. Analysis of the fluorescence excitation spectra at 77 K revealed geographical changes in the absorption.status of Chl a. Besides, the experimental results indicated that the intact needles of Pinus armandi are not ideal materials to be used in detecting the geographical variation in photochemical reaction process because the presence of thicker coat, resin etc. can conceal the spectral differences in different populations.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and Y(Z) radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S1 and S3 states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11-15 signal, were detected in Ca2+-depleted PS II. The g=11-15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S2 in Ca2+-depleted PS II. The singlet-like signal was associated with the g=11-15 signal but not with the Y(Z) (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y(Z) radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y(Z) radical was discussed.     

Two pathways of photoproduction of organic hydroperoxides on the donor side of photosystem 2 in subchloroplast membrane fragments

Earlier the catalase-insensitive formation of organic hydroperoxides (via the interaction of organic radicals produced due to redox activity of P₆₈₀ ^+· (or TyrZ^·) with molecular oxygen) has been found in Mn-depleted PS2 preparations (apo-WOC-PS2) by Khorobrykh et al. (Biochemistry 50:10658–10665, 2011). The present work describes a second pathway of the photoproduction of organic peroxides on the donor side of PS2. It was shown that illumination of CaCl₂-treated PS2 membranes (deprived of the PS2 extrinsic proteins without removal of the Mn-containing water-oxidizing complex) (CaCl₂-PS2) led to the photoproduction of highly lipophilic organic hydroperoxides (LP-OOH) (in amount corresponding to 1.5 LP-OOH per one reaction center of PS2) which significantly increased upon the addition of exogenous electron acceptor potassium ferricyanide (to 4.2 LP-OOH per one reaction center). Addition of catalase (200 U/ml) before illumination inhibited ferricyanide-induced photoproduction of hydroperoxides while no effect was obtained by adding catalase after illumination or by adding inactivated catalase before illumination. The hydroperoxide photoproduction was inhibited by the addition of exogenous electron donor for PS2, diphenylcarbazide or diuron (inhibitor of the electron transfer in PS2). The addition of exogenous hydrogen peroxide to the CaCl₂-PS2 led to the production of highly lipophilic organic hydroperoxides in the dark (3.2 LP-OOH per one reaction center). We suggest that the photoproduction of highly lipophilic organic hydroperoxides in CaCl₂-PS2 preparations occurs via redox activity of H₂O₂ produced on the donor side of PS2.     

Adaptation of photosynthesis under iron deficiency in maize

This paper explores the effects of high light stress on Fe-deficient plants. Maize (Zea mays) plants were grown under conditions of Fe deficiency and complete nutrition. Attached, intact leaves of Fe-deficient and control plants were used for gas exchange experiments under suboptimal, optimal and photoinhibitory illumination. Isolated chloroplasts were used to study photosynthetic electron transport system, compromised by the induction of Fe deficiency. The reaction centers of PS II (measured as reduction of Q, the primary electron acceptor of P 680) and PS I (measured as oxidation of P 700) were estimated from the amplitude of light induced absorbance change at 320 and 700 nm, respectively. Plants were subjected to photoinhibitory treatment for different time periods and isolated chloroplasts from these plants were used for electron transport studies. Carbon dioxide fixation in control as well as in Fe-deficient plants decreased in response to high light intensities. Total chlorophyll, P 700 and Q content in Fe-deficient chloroplasts decreased, while Chl a/b ratio and Q/P 700 ratio increased. However, electron transport through PS II suffered more after photoinhibitory treatment as compared to electron transport through PS I or whole chain. Electron transfer through PS I+PS II, excluding the water oxidation complex showed a decrease in Fe-deficient plants. However, electron transport through this part of the chain did not suffer much as a result of photoinhibition, suggesting a defect in the oxidising side of PS II.     

Functions of pheophytin, plastoquinone, iron and carotenoids in plant photosystem 2 reaction centers

Photoreduction of the intermediary electron acceptor, pheophytin (Ph), in photosystem-2 (PS-2) reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and Eh approximately -450 mV produces a narrow ESR signal of Ph. (g = 2.0033; delta H approximately 13 G) and a "doublet" centered at g = 2.00 with a splitting of 52 G at 7 K. The doublet (but not the narrow signal) is eliminated after extraction of lyophylized TSF-II with hexane, containing 0.1-0.2% methanol, or after extraction of Fe with LiClO4 and o-phenantroline, and the signal is restored by reconstitution with plastoquinone-A (PQ) or Fe++, respectively. The Fe removal results also in the development of a photoinduced ESR signal of PQ. (g approximately 2.0044; delta H approximately 9.2 G). The conclusion is made that the primary electron acceptor, Q, is in fact a complex PQ-Fe++ and that the exchange interaction of Ph. with PQ. -Fe++ is responsible for the ESR doublet. Photoreduction of Ph in TSF-IIa is accompanied by the 3-fold decrease in the formation of carotenoid triplet state (measured by the characteristic flash-induced absorbance changes) which is suggested to be a result of charge recombination in the pair [P680+ .PH.].     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

EPR signal II in cyanobacterial Photosystem II reaction-center complexes with and without the 40 kDa chlorophyll-binding subunit

The steady-state amplitude and flash-induced kinetics of EPR signal II in two Photosystem II (PS II) reaction center protein complexes from Synechococcus were measured to probe the organization of species involved in the PS II electron-transfer chain. A PS II reaction center complex (E-1) which has 47, 40, 31, 28 and 9 kDa subunits shows both fast decaying (signal II_f) and slowly decaying (signal II_s+u) EPR components. The amplitude of signal II_f, which represents Z (the donor to P-680), is about 1 spin per 30 Chl. This corresponds to one spin per reaction center in this preparation. Signal II_s+u, the slowly decaying component of signal II, reflects D, a donor to PS II on a side chain from the path of water oxidation in higher plants and algae. Signal II_s+u is present in the E-1 preparation in a ratio of about 1 spin per 40 Chl. Flash-induced signal II_f in E-1 shows biexponential decay with half-times of 20 ms and 300 ms. In a PS II reaction center complex (CP2b) which has 47, 31, 28 and 9 kDa subunits, but no 40 kDa subunit, an appreciable amount of signal II_f is observed (about 1 per 50 Chl). Less than 1 spin per 400 Chl of signal II_s+u is visible in this sample. The kinetics of Z⁺ reduction (signal II_f) in CP2b is similar to that seen in E-1 preparations, indicating that CP2b contains all of the molecules necessary for primary charge separation and secondary electron donation from Z.