Site-specific Inhibition of Photophosphorylation in Isolated Spinach Chloroplasts by Mercuric Chloride

Photophosphorylation associated with noncyclic electron transport in isolated spinach (Spinacia oleracea) chloroplasts is inhibited to approximately 50% by low concentrations of HgCl₂ (less than 1 μmole Hg²⁺/mg chlorophyll) when the electron transport pathway includes both sites of energy coupling. Reactions involving only a part of the electron transport system can give a functional isolation of at least two sites coupled to phosphorylation. Only one of these sites, located between the oxidation of plastoquinone and the reduction of cytochrome f, is sensitive to mercuric chloride. The energy conservation site located before plastoquinone and close to photosystem II is unaffected by HgCl₂ concentrations up to 10-fold those required to inhibit phosphorylation by the coupling site after plastoquinone. This site-specific inhibition may reflect a mechanistic difference in the mode of energy coupling at the two coupling sites or a variable accessibility of HgCl₂ to these sites.     

The plastoquinone pool as possible hydrogen pump in photosynthesis

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H⁺ + e^?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H⁺ uptake and the internal H⁺ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H⁺ uptake, the electron translocation through the plastoquinone pool and the internal H⁺ release (corrected for H⁺ release due to H₂O oxidation) is valid (pH_out = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H⁺ + e^?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

Biosynthesis of plastoquinone and β-carotene in isolated chloroplasts

Intact, isolated spinach chloroplasts incorporated ¹⁴C from ¹⁴CO₂ into plastoquinone and β-carotene under photosynthetic conditions. Addition of unlabelled l-tyrosine, p-hydroxyphenylpyruvate, or homogentisate increased the incorporation of ¹⁴C into plastoquinone, but decreased that into β-carotene.     

Comparative Studies on Plastoquinones. III. Distribution of Plastoquinones in Higher Plants

The distribution of plastoquinones A 45, B and C was studied in representatives from 34 different plant families beginning with liverworts and mosses to higher plants. All of these species, including many monocots and dicots, contained significant amounts of the 3 quinones. Two species of Aesculus contained plastoquinone A 20 in addition to plastoquinone A 45, B, and C. Many dicots, such as Aesculus, watermelon, tobacco and tomato accumulated increasing quantities of plastoquinones A and C₁-C₄ during the growing season. The concentrations of plastoquinones B and C₅-C₆ tended to remain at a constant low level during the season (<0.01 μmole per mg chlorophyll). Preliminary studies with bean plants (Vicia faba and Phaseolus sp.) indicate that the levels of quinones varied little under different growth conditions (day length and temp.) although Vicia faba tended to have higher PQ A values with increased temperature.     

Comparative Studies on Plastoquinones. IV. Plastoquinones in Algae

Plastoquinones A and C have been found in all classes of algae, including representatives of greens, yellow-greens, blue-greens, reds, browns and the flagellate, Euglena. Plastoquinone C from red and brown algae can be separated into 6 different types. An additional plastoquinone C has been found in Gigartina and Rhydomela. From chromatographic evidence this may be equivalent to plastoquinone C_o, a C type with a hydroxyl group on the first isoprene unit of the terpenoid sidechain of this substituted benzo-quinone. The ubiquinone, vitamin K and α-tocopherylquinone content of several algae is also reported. The presence of plastoquinone A in all green plants and many algae indicates that it may be a functional element in photosynthesis. Our study shows that plastoquinone C is more regularly present in algae than has been previously shown.     

Impact of PsbTc on forward and back electron flow, assembly, and phosphorylation patterns of photosystem II in tobacco

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn₄Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in ΔpsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in ΔpsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in ΔpsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.     

In intact leaves, the maximum fluorescence level (FM) is independent of the redox state of the plastoquinone pool: A DCMU-inhibition study

The effects of DCMU (3-(3′,4′-dichlorophenyl)-1,1-dimethylurea) on the fluorescence induction transient (OJIP) in higher plants were re-investigated. We found that the initial (F₀) and maximum (F_M) fluorescence levels of DCMU-treated leaves do not change relative to controls when the treatment is done in complete darkness and DCMU is allowed to diffuse slowly into the leaves either by submersion or by application via the stem. Simultaneous 820 nm transmission measurements (a measure of electron flow through Photosystem I) showed that in the DCMU-treated samples, the plastoquinone pool remained oxidized during the light pulses whereas in uninhibited leaves, the F_M level coincided with a fully reduced electron transport chain. The identical F_M values with and without DCMU indicate that in intact leaves, the F_M value is independent of the redox state of the plastoquinone pool. We also show that (i) the generally observed F₀ increase is probably due to the presence of (even very weak) light during the DCMU treatment, (ii) vacuum infiltration of leaf discs leads to a drastic decrease of the fluorescence yield, and in DCMU-treated samples, the F_M decreases to the I-level of their control (leaves vacuum infiltrated with 1% ethanol), (iii) and in thylakoid membranes, the addition of DCMU lowers the F_M relative to that of a control sample.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes

Pyrene fluorescence quenching by plastoquinone was used to estimate the rate of plastoquinone lateral diffusion in soybean phosphatidylcholine proteoliposomes containing the following integral membrane proteins: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc₁, and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 · 10^-7 cm² s^-1 in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc₁, and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration.     

Realtime kinetics of the light driven steps of photosynthetic water oxidation in living organisms by “stroboscopic” fluorometry

We develop a rapid “stroboscopic” fluorescence induction method, using the fast repetition rate fluorometry (FRRF) technique, to measure changes in the quantum yield of light emission from chlorophyll in oxygenic photosynthesis arising from competition with primary photochemical charge separation (P₆₈₀* ➔ P₆₈₀⁺Q_A⁻). This method determines the transit times of electrons that pass through PSII during the successive steps in the catalytic cycle of water oxidation/O₂ formation (S states) and plastoquinone reduction in any oxygenic phototroph (in vivo or in vitro). We report the first measurements from intact living cells, illustrated by a eukaryotic alga (Nannochloropsis oceanica). We demonstrate that S state transition times depend strongly on the redox state of the PSII acceptor side, at both Q_B and the plastoquinone pool which serve as the major locus of regulation of PSII electron flux. We provide evidence for a kinetic intermediate S3′ state (lifetime 220 μs) following formation of S3 and prior to the release of O₂. We compare the FRRF-detected kinetics to other previous spectroscopic methods (optical absorbance, EPR, and XES) that are applicable only to in vitro samples.     

Sites of inhibition by disulfiram in thylakoid membranes

Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron transport in broken chloroplasts. A major site of inhibition is detected on the electron-acceptor side of photosystem II between Q_A, the first plastoquinone electron-acceptor, and the second plastoquinone electron-acceptor, Q_B. This site of inhibition is shown by a severalfold increase in the half-time of Q⁻_A oxidation, as monitored by the decay of the variable chlorophyll a flourescence after an actinic flash. Another site of inhibition is detected in the functioning of the reaction center of photosystem II; disulfiram is observed to quench the room temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from H₂O to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected in the partial reactions associated with photosystem I.     

Requirement for plastoquinone a in the hill reaction of isolated chloroplasts

In order to avoid the problem of an uncoupling effect in stimulation of potential Hill reaction activity during extraction with heptane. Hill reaction activity was measured with uncoupled spinach chloroplasts. The reactions were also run under aerobic conditions. Under these conditions a correction for a 3-(p-chlorophenyl)-1, 1-dimethylurea insensitive, photostimulated O₂ uptake reaction is necessary, especially with extracted chloroplasts. The reaction displayed proper Hill reaction stoichiometry in untreated and restored systems if these corrections were made. Plastoquinone A always restored some activity to the extracted chloroplasts but never restored activity to the original level. Additional activity could be restored by the crude extract. It appears that there is a clear requirement for plastoquinone A in the Hill reaction but the restoration of maximum activity depends upon additional factors. The variability of response to plastoquinone A which has been reported in various laboratories may be determined by the degree of extraction of the other materials.     

The cationic plastoquinone radical of the chloroplast water splitting complex: Hyperfine splitting from a single methyl group determines the EPR spectral shape of Signal II

The proposal that EPR Signal II in spinach chloroplasts is due to a plastoquinone cation radical (O'Malley, P.J. and Babcock, G.T. (1983) Biophys. J. 41, 315a) has been investigated in further detail. The similarity in spectral shape between Signal II and the 2-methyl-5-isopropylhydroquinone cation radical is shown to arise from hyperfine coupling to one methyl group for both radicals. A well-resolved four line EPR spectrum of approximate relative intensity 1:3:3:1 for membrane orientation parallel and perpendicular to the applied magnetic field direction also indicates that the partially resolved structure of Signal II is due to hyperfine interaction with one methyl group, i.e., the 2-CH₃ group of the plastoquinone cation radical. The ENDOR band observed for this coupling is similar to that observed for methyl group bands of model quinone radicals. The principal hyperfine tensor values obtained for the methyl group interactions are A_⊥ = 27.2 MHz and $\text{A}{}_{\mathrm{}}\text{= 31.4}\text{MHz}$. The large isotropic coupling value (28.6 MHz) of the plastoquinone cation radical's 2-methyl group in vivo indicates that the antisymmetric orbital is the sole contributor to the spin-density distribution of Signal II. The orientation data also suggest that the plastoquinone cation radical is oriented such that the C-CH₃ bond direction, and hence the aromatic ring plane, lies perpendicular to the membrane plane.     

Changes in Photosystem II Account for the Circadian Rhythm in Photosynthesis in Gonyaulax polyedra

Cell-free extracts that show activity in photosynthetic electron flow have been prepared from the unicellular dinoflagellate, Gonyaulax polyedra. Electron flow, as O₂ uptake, was measured through both photo-system I and II from water to methyl viologen, through photosystem I alone from reduced 2,6-dichlorophenol indophenol to methyl viologen which does not include the plastoquinone pool or from duroquinol to methyl viologen which includes the plastoquinone pool. Electron flow principally through photosystem II was measured from water to diaminodurene and ferricyanide, as O₂ evolution. Cultures of Gonyaulax were grown on a 12-hour light:12 hour dark cycle to late log phase, then transferred to constant light at the beginning of a light period. After 3 days, measurements of electron flow were made at the maximum and minimum of the photosynthetic rhythm, as determined from measurements of the rhythm of bioluminescence. Photosynthesis was also measured in whole cells, either as ¹⁴C fixation or O₂ evolution. Electron flow through both photosystems and through photosystem II alone were clearly rhythmic, while electron flow through photosystem I, including or excluding the plastoquinone pool, was constant with time in the circadian cycle. Thus, only changes in photosystem II account for the photosynthesis rhythm in Gonyaulax.     

Kinetics of photosystem II electron transport: a mathematical analysis based on chlorophyll fluorescence induction

The OJDIP rise in chlorophyll fluorescence during induction at different light intensities was mathematically modeled using 24 master equations describing electron transport through photosystem II (PSII) plus ordinary differential equations for electron budgets in plastoquinone, cytochrome f, plastocyanin, photosystem I, and ferredoxin. A novel feature of the model is consideration of electron in- and outflow budgets resulting in changes in redox states of Tyrosine Z, P680, and Q_A as sole bases for changes in fluorescence yield during the transient. Ad hoc contributions by transmembrane electric fields, protein conformational changes, or other putative quenching species were unnecessary to account for primary features of the phenomenon, except a peculiar slowdown of intra-PSII electron transport during induction at low light intensities. The lower than F _m post-flash fluorescence yield F _f was related to oxidized tyrosine Z. The transient J peak was associated with equal rates of electron arrival to and departure from Q_A and requires that electron transfer from Q_A ^? to Q_B be slower than that from Q_A ^? to Q_B ^?. Strong quenching by oxidized P680 caused the dip D. Reduced plastoquinone, a competitive product inhibitor of PSII, blocked electron transport proportionally with its concentration. Electron transport rate indicated by fluorescence quenching was faster than the rate indicated by O₂ evolution, because oxidized donor side carriers quench fluorescence but do not transport electrons. The thermal phase of the fluorescence rise beyond the J phase was caused by a progressive increase in the fraction of PSII with reduced Q_A and reduced donor side.     

Activation of a Reserve Pool of Photosystem II in Chlamydomonas reinhardtii Counteracts Photoinhibition

The effect of strong irradiance (2000 micromole photons per square meter per second) on PSII heterogeneity in intact cells of Chlamydomonas reinhardtii was investigated. Low light (LL, 15 micromole photons per square meter per second) grown C. reinhardtii are photoinhibited upon exposure to strong irradiance, and the loss of photosynthetic functioning is due to damage to PSII. Under physiological growth conditions, PSII is distributed into two pools. The large antenna size (PSII_α) centers account for about 70% of all PSII in the thylakoid membrane and are responsible for plastoquinone reduction (Q_b-reducing centers). The smaller antenna (PSII_β) account for the remainder of PSII and exist in a state not yet able to photoreduce plastoquinone (Q_b-nonreducing centers). The exposure of C. reinhardtii cells to 60 minutes of strong irradiance disabled about half of the primary charge separation between P680 and pheophytin. The PSII_β content remained the same or slightly increased during strong-irradiance treatment, whereas the photochemical activity of PSII_α decreased by 80%. Analysis of fluorescence induction transients displayed by intact cells indicated that strong irradiance led to a conversion of PSII_β from a Q_b-nonreducing to a Q_b-reducing state. Parallel measurements of the rate of oxygen evolution revealed that photosynthetic electron transport was maintained at high rates, despite the loss of activity by a majority of PSII_α. The results suggest that PSII_β in C. reinhardtii may serve as a reserve pool of PSII that augments photosynthetic electron-transport rates during exposure to strong irradiance and partially compensates for the adverse effect of photoinhibition on PSII_α.     

Na-Dithionite Promotes Photosynthetic Sulfide Utilization by the Cyanobacterium Oscillatoria limnetica

The light- and sulfide-dependent induction process leading to photosystem I-mediated sulfide utilization by Oscillatoria limnetica, for either H₂ evolution or CO₂ photoassimilation, was studied. The identical dependence on pH of the lag length, the inhibition of leucine incorporation and final H₂S concentration imply that the latter exerts a deleterious effect on nonadapted cells.

Na-dithionite (Na₂S₂O₄), Na-sulfite (Na₂SO₃), or ethanol cannot serve as photosynthetic electron donors. However, when these compounds were added to the sulfide-containing system, the need for induction was eliminated. At pH 6.9, in the presence of 3.5 millimolar sulfide, these substances (at concentrations of 10 millimolar, 5 millimolar, or 0.4 molar, respectively) completely abolished the delay preceding sulfide-dependent H₂ evolution. It is suggested that all three compounds expose a site capable of directly accepting sulfide electrons.

Only dithionite could adapt the cells to sulfide utilization on its own. Sulfite or ethanol acted only in the presence of sulfide. It is implied that this specific activity of dithionite is related to its characteristic low redox potential.

Sulfide-dependent H₂ evolution was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but was inhibited by the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, in the presence as well as in the absence of dithionite. In both cases, therefore, the plastoquinone was implied in the electron transport from sulfide.

     

Activation of Chlororespiration Increases Chlorophyll Fluorescence Yield in Chlorella Adapted to Darkness at High Temperature

A tenfold increase in chlororespiration during dark incubation of Chlorella perynoidosa Chick CALU-175 at high temperature doubled the initial chlorophyll fluorescence yield (F ₀). The presence of iodacetamide or unmetabolizable glucose analog 2-deoxy-D-glucose prevented increase in both chlororespiration and F ₀ yield. The rates of chlororespiration and F ₀ yield growth demonstrated a similar pattern of temperature dependence. Inhibition of electron transport between Q _A and plastoquinone with diuron prevented increase in F ₀ during dark incubation of the cells at high temperature. Apparently, a pool of plastoquinone was restored in the chlororespiratory chain during the dark incubation at 37.5–41°C, and plastoquinone exchanged electrons with Q _A. This is the cause of Q _A reduction and subsequent increase in F ₀ yield.