Plastoquinone Research Articles

Changes in quinone profiles of hot spring microbial mats with a thermal gradient

The respiratory and photosynthetic quinones of microbial mats which occurred in Japanese sulfide-containing neutral-pH hot springs at different temperatures were analyzed by spectrochromatography and mass spectrometry. All of the microbial mats that developed at high temperatures (temperatures above 68 degreesC) were so-called sulfur-turf bacterial mats and produced methionaquinones (MTKs) as the major quinones. A 78 degreesC hot spring sediment had a similar quinone profile. Chloroflexus-mixed mats occurred at temperatures of 61 to 65 degreesC and contained menaquinone 10 (MK-10) as the major component together with significant amounts of either MTKs or plastoquinone 9 (PQ-9). The sunlight-exposed biomats growing at temperatures of 45 to 56 degreesC were all cyanobacterial mats, in which the photosynthetic quinones (PQ-9 and phylloquinone) predominated and MK-10 was the next most abundant component in most cases. Ubiquinones (UQs) were not found or were detected in only small amounts in the biomats growing at temperatures of 50 degreesC and above, whereas the majority of the quinones of a purple photosynthetic mat growing at 34 degreesC were UQs. A numerical analysis of the quinone profiles was performed by using the following three parameters: dissimilarity index (D), microbial divergence index (MDq), and bioenergetic divergence index (BDq). A D matrix tree analysis showed that the hot spring mats consisting of the sulfur-turf bacteria, Chloroflexus spp., cyanobacteria, and purple phototrophic bacteria formed distinct clusters. Analyses of MDq and BDq values indicated that the microbial diversity of hot spring mats decreased as the temperature of the environment increased. The changes in quinone profiles and physiological types of microbial mats in hot springs with thermal gradients are discussed from evolutionary viewpoints.

Ultraviolet-B Induced Damage to Photosystem II in Intact Filaments of Spirulina platensis

Abstract When Spirulina platensis filaments were exposed to 0.75 mW.m-2.s-1 of ultraviolet-B radiation (the ultraviolet-B radiation under clear sky condition is ~1.0 mW.m-2.s-1), an inhibition in photosystem II activity was observed, the inhibition being 90% after 90 min exposure. Upon exposure to ultraviolet-B, the room temperature emission characteristics of Spirulina cells were altered when excited with light primarily absorbed by chlorophyll a or phycobilisomes. When the cells were exposed for 3 h the emission at 685 nm (F685), when excited at 440 nm (primarily chlorophyll a absorption), was enhanced compared to 715 nm (F 715) band of photosystem I suggesting a decrease in energy transfer from photosystem II to photosys­ tem I. Similarly, when the cells were excited at 580 nm (primarily the phycobilisomes), the ratio of emission intensity at 685 nm (F685) to that of 655 nm (F655) was decreased in the exposed cells. This change in emission characteristics seems to be linked with the uncoupling of the energy transfer from allophycocyanin to chlorophyll a of photosystem II. A small shift in emission peak positions was also indicated when excited either at 440 nm or 580 nm. Analysis of the fast induction of chlorophyll a transients in the presence and absence of 10 μm 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) indicated that ultraviolet-B expo­ sure initially affects Qᴀ, the primary stable acceptor of photosystem II, and then the plastoquinone (PQ) pool. Our results on the loss in photosystem Il-catalyzed Hill activity with p-benzoquinone or dichlorobenzoquinone as electron acceptors also supports the contention that ultraviolet-B, even at low dose, initially alters the Qᴀ of photosystem II and subsequently PQ pool. The analysis of functional pool size of Spirulina suggests a substantial decrease in the functional pool size after 2 h UV-B exposure. These results indicate that in Spirulina low intensity of ultraviolet-B initially damages the reaction centre of photosystem II.

Reduction of the plastoquinone pool by exogenous NADH and NADPH in higher plant chloroplasts: Characterization of a NAD(P)H–plastoquinone oxidoreductase activity

Chlorophyll fluorescence measurements were performed on osmotically lysed potato chloroplasts in order to characterize the reactions involved in the dark reduction of photosynthetic inter-system chain electron carriers. Addition of NADH or NADPH to lysed chloroplasts increased the chlorophyll fluorescence level measured in the presence of a non-actinic light until reaching F max, thus indicating an increase in the redox state of the plastoquinone (PQ) pool. The fluorescence increase was more pronounced when the experiment was carried out under anaerobic conditions and was about 50% higher when NADH rather than NADPH was used as an electron donor. The NAD(P)H–PQ oxidoreductase reaction was inhibited by diphenylene iodonium, N-ethylmaleimide and dicoumarol, but insensitive to rotenone, antimycin A and piericidin A. By comparing the substrate specificity and the inhibitor sensitivity of this reaction to the properties of spinach ferredoxin–NADP +-reductase (FNR), we infer that FNR is not involved in the NAD(P)H–PQ oxidoreductase activity and conclude to the participation of rotenone-insensitive NAD(P)H–PQ oxidoreductase. By measuring light-dependent oxygen uptake in the presence of DCMU, methyl viologen and NADH or NADPH as an electron donors, the electron flow rate through the NAD(P)H–PQ oxidoreductase is estimated to about 160 nmol O 2 min −1 mg −1 chlorophyll. The nature of this enzyme is discussed in relation to the existence of a thylakoidal NADH dehydrogenase complex encoded by plastidial ndh genes.

Temporal Changes in State Transitions and Photosystem Organization in the Unicellular, Diazotrophic Cyanobacterium Cyanothece sp. ATCC 51142.

The unicellular Cyanobacterium Cyanothece sp. ATCC 51142, grown under alternating 12-h light/12-h dark conditions, temporally separated N2 fixation from photosynthesis. The regulation of photosynthesis was studied using fluorescence spectra and kinetics to determine changes in state transitions and photosystem organization. The redox poise of the plastoquinone (PQ) pool appeared to be central to this regulation. Respiration supported N2 fixation by oxidizing carbohydrate granules, but reduced the PQ pool. This induced state 2 photosystem II monomers and lowered the capacity for O2 evolution. State 2 favored photosystem I trimers and cyclic electron transport, which could stimulate N2 fixation; the stimulation suggested an ATP limitation to N2 and CO2 fixation. The exhaustion of carbohydrate granules at around 6 h in the dark resulted in reduced respiratory electron flow, which led to a more oxidized PQ pool and produced a sharp transition from state 2 to state 1. This transient state 1 returned to state 2 in the remaining hours of darkness. In the light phase, photosystem II dimerization correlated with increased phycobilisome coupling to photosystem II (state 1) and increased rates of O2 evolution. However, dark adaptation did not guarantee state 2 and left photosystem I centers in a mostly monomeric state at certain times.

The discovery and structural requirements of inhibitors of p-hydroxyphenylpyruvate dioxygenase

The benzoylcyclohexane-1,3-diones, the triketones, are potent bleaching herbicides whose structure-activity relationships and physical properties are substantially different from classical bleaching herbicides, which affect phytoene desaturase. The first clue to their unique mechanism of action was the discovery that rats treated with a triketone were found to be tyrosinemic. Additionally, examination of the rat urine revealed the accumulation of p-hydroxyphenylpyruvate (HPP) and p-hydroxyphenyllactate. These results suggested that this chemically induced tyrosinemia was the result of the inhibition of p-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), and this suggestion was confirmed when a triketone was shown to be a potent inhibitor of rat liver HPPD. In plants, HPPD is a component of the biosynthetic pathway to plastoquinone (PQ), which in turn is a key cofactor of phytoene desaturase. The expectation that triketone-treated plants should accumulate tyrosine while having reduced PQ levels was dramatically demonstrated in the meristematic tissue of ivyleaf morningglory. Plant HPPD, like the mammalian enzyme, was inhibited in vitro by triketones. These biochemical effects provide evidence that the triketone herbicidal mechanism of action is HPPD inhibition leading to a deficiency of PQ, a key cofactor for carotenoid biosynthesis. Other chemical classes of bleaching herbicides were also examined for their ability to elevate tyrosine and deplete PQ as a definitive means of establishing their mode of action and for delineating the structural and physical chemical requirements for an HPPD herbicide. Evidence is provided to support the claim that a 2-benzoylethen-1-ol substructure is the minimum substructure required for a potent HPPD inhibitor.

Electron paramagnetic resonance and mutational analyses revealed the involvement of photosystem II-L subunit in the oxidation step of Tyr-Z by P680+ to form the Tyr-Z+P680Pheo- state in photosystem II.

To reveal the molecular mechanism of involvement of photosystem II (PSII)-L protein in the electron transfer in PSII, effects of mutations in PSII-L on the photochemistry of PSII were investigated by means of electron paramagnetic resonance (EPR) and flash photolysis. Wild type and a series of mutant versions of PSII-L were overproduced in Escherichia coliand chromatographically purified. Plastoquinone 9 (PQ-9) depleted PSII reaction center core complex consisting of CP47/D1/D2/Cytb-559/PSII-I/PSII-W was prepared and reconstituted with the wild type and each mutant version of PSII-L together with or without PQ-9. EPR signal indicating the formation of Tyr-Z+P680Pheo- state upon room-temperature illumination disappeared in CP47/D1/D2/Cytb-559/PSII-I/PSII-W, and it was recovered when the complex was reconstituted with the wild-type PSII-L. Mutation of a few amino acid residues in the carboxyl-terminal region of PSII-L, such as substitution of a triad of Tyr34, Phe35, and Phe36 by Leu, selectively resulted in the loss of the capability of PSII-L to recover the light-induced formation of Tyr-Z+P680Pheo- state in the reconstituted complex. Hydropathy profile of PSII-L suggests that it spans the membrane once by a hydrophobic stretch of the carboxyl-terminal side as its carboxyl end to face to the lumen. If this is the case, the amino acid residues essential for PSII-L to function are expected to be located close to the donor side of P680, suggesting the interaction of PSII-L with Tyr-Z (and/or Tyr-D) or P680 to facilitate the oxidation of Tyr-Z by P680+ to form Tyr-Z+P680Pheo- state in PSII. Evidence against PSII-L being involved in the electron transfer from Pheo- to QA was obtained by the flash photolysis experiments.

Kinetics of S2 and S3 reduction by tyrosine Y(D) and other endogenous donors as a function of temperature in spinach PS II membrane fragments with a reconstituted plastoquinone pool.

The characteristic period four oscillation patterns of oxygen evolution induced by a train of single-turnover flashes were measured as a function of temperature in dark-adapted photosystem II (PS II) membrane fragments that were reconstituted with native plastoquinone-9 (PQ-9) by a recently developed procedure [Kurreck, J., Seeliger, A. G., Reifarth, F., Karge, M., & Renger, G. (1995) Biochemistry 34, 15721-15731]. The following results were obtained: (a) within the range 0-35 degrees C, the probabilities of misses (alpha) and double-hits (beta) and the dark population of redox state S1 exhibit similar dependencies on the temperature; (b) below a characteristic temperature theta(c) these parameters remain virtually independent of temperature, above theta(c) (theta(c) = 20 degrees C for alpha and beta; theta(c) = 30 degrees C for S1) the values of alpha and beta increase whereas S1 decreases; and (c) the dark decay of S2 and S3 via fast and slow kinetics owing to reduction of the water oxidase by Y(D) and other endogenous electron donor(s), respectively, exhibits comparatively strong temperature dependencies with the following activation energies: E(A)(S2(fast)) = 60 +/- 10 kJ/mol, EA(S3(fast)) = 55 +/- 10 kJ/mol, E(A)(S2(slow)) = 80 +/- 5 kJ/mol, and E(A)(S3(slow)) = 75 +/- 5 kJ/mol. These values of PQ-9 reconstituted PS II membrane fragments are very similar to those that were previously reported for thylakoids [Messinger, J., Schröder, W. P., & Renger, G. (1993) Biochemistry 32, 7658-7668]. These findings reveal that the reaction coordinates of feeding electrons by endogenous electron donors into the water oxidizing complex (WOC) that attains the redox states S2 and S3 is virtually invariant to Triton X-100 treatment used in the isolation procedure of PS II membrane fragments from thylakoids. Implications of these findings are discussed.

Role of PSII-L protein (psbL gene product) on the electron transfer in photosystem II complex. 1. Over-production of wild-type and mutant versions of PSII-L protein and reconstitution into the PSII core complex.

To establish a system for over-production of PSII-L protein which is a component of photosystem II (PSII) complex, a plasmid designated as pMAL-psbL was constructed and expressed in Escherichia coli JM109. A fusion protein of PSII-L and maltose-binding proteins (53 kDa on SDS-PAGE) was accumulated in E. coli cells to a level of 10% of the total protein upon isopropyl-beta-D-thiogalactopyranoside (IPTG) induction. The carboxyl-terminal part of 5.0 kDa was cleaved from the fusion protein and purified by an anion exchange column chromatography in the presence of detergents. This 5.0 kDa protein was identified as PSII-L by amino-terminal amino acid sequence analysis and the chromatographic behavior on an anion exchange gel. A few types of mutant PSII-L were also prepared by the essentially same procedure except for using plasmids which contain given mutations in psbL gene. Plastoquinone-9 (PQ-9) depleted PSII reaction center core complex consisting of D1, D2, CP47, cytochrome b-559 (cyt b-559), PSII-I and PSII-W was reconstituted with PQ-9 and digalactosyldiglyceride (DGDG) together with the wild-type or mutant PSII-L produced in E. coli or isolated PSII-L from spinach. Significant difference between the wild-type PSII-L proteins from E. coli and spinach was not recognized in the effectiveness to recover the photo-induced electron transfer activity in the resulting complexes. The analysis of stoichiometry of PQ-9 per reaction center in the PQ-9 reconstituted PS II revealed that two molecules of PQ-9 were reinserted into a reaction center independent of the presence or absence of PSII-L. These results suggest that PSII-L recovers the electron transfer activity in the reconstituted RC by a mechanism different from the stabilization of PQ-9 in the Q(A) site of PSII. Ubiquinone-10 (UQ-10), but not plastoquinone-2 (PQ-2), substituted PQ-9 for recovering the PSII-L supported electron transfer activity in the reconstituted PSII reaction center complexes. The results obtained with the mutant PSII-L proteins revealed that the carboxyl terminal part rather than amino terminal part of PSII-L is crucial for recovering the electron transfer activity in the reconstituted complexes.

Mechanism of Electron Flow through the QB Site in Photosystem II. 4. Reaction Mechanism of Plastoquinone Derivatives at the QB Site in Spinach Photosystem II Membrane Fragments

Interactions of externally added plastoquinone (PQ) derivatives (PQ0-PQ3) with the photosystem II (PSII) acceptor side were investigated in PSII membrane fragments prepared from spinach by measuring the photoreduction rates of PQ derivatives at various PQ concentrations, and the following results were obtained. From the kinetic analysis, all the PQ derivatives (PQ0-PQ3) except PQ3 were shown to accept electrons at two sites (the QB site and the PQ site) as in the case of Synechococcus vulcanus PSII particles with benzoquinone derivatives [Satoh et al. (1995) Plant Cell Physiol. 36: 597]. Affinities of PQ derivatives at the QB site increased as the length of the isoprene side chain got longer, while those at the PQ site were not very much different for all the PQ derivatives tested in this study. The inhibitory effect of DCMU was noncompetitive, and, therefore, the affinity of PQ3 for the PQ site was determined while that for the QB site could not be estimated presumably due to its fairly high affinity to the site. Based on the results obtained using PQ derivatives, the mechanism of interaction of an authentic PQ, PQ9, at the QB site is discussed.

Mechanisms of Electron Flow through the QB Site in Photosystem II. 3. Effects of the Presence of Membrane Structure on the Redox Reactions at the QB Site

Reduction kinetics of chloro- or methyl-substi tuted benzoquinones (BQs) in spinach PSII membrane fragments or /!-heptyl-/7- D-thioglucoside-extracted PSII core complexes (HTG-PSII) was studied and compared to that in cyanobacterial PSII core complexes [Satoh et al. (1995) Plant Cell Physiol. 36: 597]. It was found that the BQs accept electrons at two sites (the QB and PQ sites) in the both spinach preparations as in the cyanobacterial preparation. Maximum turnover rates (Vmax) and binding affinities (Km) of the two sites were estimated. Comparison of the values in PSII membrane fragments with those in HTG-PSII showed that removal of the membrane structure or lightharvesting chlorophyll a/b protein complexes from the PSII core complexes had little effect on the characteristics of the Q,, site, indicating that the HTG-PSII have the intact QB site and are good materials to study the site. The Km and Vmn values were comparable to those in the cyanobacterial preparation. Low affinity (high Km values) and high Vmtx values of methyl-substituted BQs to the QB site and almost the same rate of intrinsic electron flow through the QB site in the both spinach preparations further support the hypothesis that the plastoquinone (PQ) molecule at the QB site is not replaced by another PQ molecule but, after reduction, only its head group goes out of the QB pocket, and comes back after transferring the electrons and protons to a free PQ molecule [Satoh et al. (1993) Z. Naturforsch. 48c: 174].

Influence of the redox state of ubiquinones and plastoquinones on the order of lipid bilayers studied by fluorescence anisotropy of diphenylhexatriene and trimethylammonium diphenylhexatriene

The measurements of diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) fluorescence anisotropy in egg yolk lecithin (EYL) and of DPH anisotropy in dipalmitoylphosphatidylcholine (DPPC) liposomes containing different concentrations of oxidized and reduced ubiquinone (UQ) and plastoquinone (PQ) homologues have been performed. All the oxidized UQ homologues strongly induced ordering of EYL membrane structure, whereas in DPPC liposomes, above the phase transition temperature, the most pronounced effect showed UQ-4. PQ-2 and PQ-9 were less effective than the corresponding ubiquinones in this respect. The reduced forms of UQ and PQ homologues increased the order of membrane lipids to a smaller extent than the corresponding quinones both in the interior of the membrane and closer to its surface. Nevertheless, the investigated prenylquinols showed stronger increase in the membrane order than α-tocopherol or α-tocopherol acetate, which could be connected with binding of prenylquinol head groups to phospholipid molecules by hydrogen bonds. The strong ordering influence of ubiquinones on the membrane structure was attributed to methoxyl groups of the UQ quinone rings.

Mechanism of Electron Flow through the Q<sub>B</sub> Site in Photosystem II. 1. Kinetics of the Reduction of Electron Acceptors at the Q<sub>B</sub> and Plastoquinone Sites in Photosystem II Particles from the Cyanobacterium <italic>Synechococcus vulcanus</italic>

Various derivatives of benzoquinone (BQ) were found to be reduced at two sites [the QB and plastoquinone (PQ) sites] in photosystem II particles from Synechococcus vulcanus, and the relationship between the structures of BQs and the kinetics of such reduction at each site were studied. Affinities of BQs for both the QB and the PQ site and the maximum turnover rates at the two sites were estimated by computer simulation of the dependence on the concentration of the BQ of the rate of oxygen evolution. Affinities of BQs for the QB sites determined by this method agreed well with those determined from competition between BQs and DCMU for the QB site. All methyl-substituted BQs had low affinities for the QB site, and tetramethyl-p-benzoquinone had almost no affinity. An increase in the number of chlorine atoms in the quinone ring increased the affinity, and the position of such substitutions had a great effect on the affinity when two positions on the ring were occupied by two chlorine atoms or methyl groups. The affinities of BQs for the PQ site were almost the same for all BQs tested in this experiment but the maximum turnover rates at this site varied greatly from one derivative to another. The results are consistent with the hypothesis that the binding of the PQ molecule to the QB site is attributable not to its quinone ring but to its isoprenoid chain and, moreover, that the electron transfer through the QB site occurs not by replacement of the PQ molecule but by donation of electrons to another PQ molecule.

Erratum: Semiempirical quantum chemical treatment of the standard reduction potentials of quinone and plastoquinone in water

The standard electrode potential for the quinone (Q)-hydroquinone (QH2) couple in aqueous acidic media has been explicitly calculated. Molecular geometries of Q and QH2 have been optimized. Protonation of Q, i.e., the formation of QH+ and QH, have been considered. Molecular geometries of these species have been thoroughly optimized. The energy of complexation of these molecules with water have been calculated by optimizing the structures of the hydrated complexes Q · 6H2O, QH2 · 6H2O, QH+ · 6H2O. and QH · 6H2O. The ion–solvent interaction energy of QH+ · 6H2O, in turn, has been calculated by considering the complex QH+ · 6H2O… 2H2O, where the two extra water molecules approach the charge center of the complex QH+ · 6H2O vertically from top and bottom of the quinonoid ring. The standard reduction potential calculated by the CNDO method, 0.8548 V, is somewhat larger than the experimental potential, 0.6998 V, at 25°C. But the INDO value, 0.7085 V, is in excellent agreement with the observed potential. The electrode potential for the plastoquinone (PQ)-plastohydroquinone (PQH2) couple present in the aqueous pool in chloroplast has been calculated by the INDO method. The basic geometries of PQ, PQH+, and PQH2/sb have been synthesized by adopting the optimized geometries of Q, QH+, and QH2 and considering methyl substituents as well as an isoprenoid side chain containing up to 3 isoprene units with possible geometrical isomerism. The hydrated species PQ · 6H2O, PQH+ · 6H2O, and PQH2 · 6H2O are unstable compared to the isolated species PQ, PQH+, and PQH2, respectively. In fact, we have found that the hydration of PQH+ and PQH2 is much less extensive, and stability arises only when the hydroxyl groups in these two molecules are hydrogen-bonded to water molecules. But PQH+ is also stabilized through the association of two more water molecules in the vertical direction. For this reason, we have calculated the reduction potential of the PQ/PQH2 system from the energies of the isolated molecules PQH2 and the hydrated species PQH+ · 2H2O. The computed standard reduction potential is 0.2785 V and it yields a potential of 0.07V at pH 7 at 25°C, which is in good agreement with the reduction potential 0.11 V observed for plastoquinone in the aqueous pool in chloroplast. © 1994 John Wiley & Sons, Inc.

Blue Light-Enhanced Photosynthetic Oxygen Evolution from Liposome-Bound Photosystem II Particles; Possible Role of the Xanthophyll Cycle in the Regulation of Cyclic Electron Flow Around Photosystem II?

Abstract Light-driven electron transport in liposome-bound photosystem II (PS-II) particles be­tween water and ferricyanide was monitored by bare platinum electrode oxymetry. The modi­fication of the experimental system with the exogenous quinones α-tocopherol quinone ( α-TQ) or plastoquinone (PQ) resulted in a pronounced effect on photosynthetic oxygen evolution. The presence of α-tocopherolquinone ( α-TQ) in PS-II samples decreased the rate of red light-induced oxygen evolution but increased the rate of green light-induced oxygen evolution. Blue light applied to the assay system in which oxygen evolution was saturated by red light resulted in a further increase of the oxygen signal. These findings are interpreted in terms of a cyclic electron transport around PS-II, regulated by an excitation state of β-carotene in the reaction centre of PS-II. A mechanism is postulated according to which energetic coupling of β-carotene in the reaction centre of PS-II and that of other antenna carotenoid pigments is regulated by the portion of the xanthophyll violaxanthin, which is under control of the xanthophyll cycle.

EPR and ENDOR investigation of the primary electron acceptor radical anion QA.- in iron-depleted photosystem II membrane fragments.

Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe2+ associated with the quinones QA and QB in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to those of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents. Q-band EPR yielded the principal values of the g-tensor for PQ-9.- in 2-propanol and QA.- in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9.- in vitro and on QA.- in the iron-depleted PS II samples. For the former a complete set of isotropic 1H hyperfine coupling constants and hyperfine tensors of the two methyl groups and the alpha-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. 1H-ENDOR spectra of QA.- in iron-depleted PS II samples have been measured in buffers made in H2O and D2O. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of QA and specific protein residues. The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.

POLYPHASIC CHLOROPHYLL a FLUORESCENCE TRANSIENT IN PLANTS AND CYANOBACTERIA*

Abstract— The variable chlorophyll (Chl) a fluorescence yield is known to be related to the photochemical activity of photosystem II (PSII) of oxygen‐evolving organisms. The kinetics of the fluorescence rise from the minimum yield, F0, to the maximum yield, Fm, is a monitor of the accumulation of net reduced primary bound plastoquinone (QA) with time in all the PSII centers. Using a shutter‐less system (Plant Efficiency Analyzer, Hansatech, UK), which allows data accumulation over several orders of magnitude of time (40 μs to 120 s), we have measured on a logarithmic time scale, for the first time, the complete polyphasic fluorescence rise for a variety of oxygenic plants and cyanobacteria at different light intensities. With increasing light intensity, the fluorescence rise is changed from a typical O‐I‐P characteristic to curves with two intermediate levels J and I, both of which show saturation at high light intensity but different intensity dependence. Under physiological conditions, Chl a fluorescence transients of all the organisms examined follow the sequence of O‐J‐I‐P. The characteristics of the kinetics with respect to light intensity and temperature suggest that the O‐J phase is the photochemical phase, leading to the reduction of QA to QA‐. The intermediate level I is suggested to be related to a heterogeneity in the filling up of the plastoquinone pool. The P is reached when all the plastoquinone (PQ) molecules are reduced to PQH2. The addition of 3‐(3–4‐dichlorophenyl)‐1,1‐dimethylurea leads to a transformation of the O‐J‐I‐P rise into an O‐J rise. The kinetics of O‐J‐I‐P observed here was found to be similar to that of O‐I1‐I2‐P, reported by Neubauer and Schreiber (Z. Naturforsch.42c, 1246–1254, 1987). The biochemical significance of the fluorescence steps O‐J‐I‐P with respect to the filling up of the plastoquinone pool by PSII reactions is discussed.

L protein, encoded by psbL, restores normal functioning of the primary quinone acceptor, Q A, in isolated D1/D2/CP47/Cytb-559/I photosystem II reaction center core complex

Plastoquinone-9(PQ-9)-depleted PSII reaction center core complex, consisting of CP47/D1/D2/Cytb-55911, was isolated from spinach PSII particles. PQ-9, lipids and several proteins were extracted from the original PSII particles and separated by several steps of chromatography to be reconstituted into the isolated complex. PQ-9 reconstituted in the complex with the help of thylakoid lipids (digalactosyldiglyceride) did not function as Q A by itself. However, PQ-9 simultaneously reconstituted with L protein and the thylakoid lipids successfully functioned as Q A in the complex. Other proteins of PSII origin, such as CP43, H, K, nuclear encoded 4.1 and 5.0 kDa proteins, are unable to restore the Q A activity in the complex.

Semiempirical quantum chemical treatment of the standard reduction potentials of quinone and plastoquinone in water

AbstractThe standard electrode potential for the quinone (Q)‐hydroquinone (QH2) couple in aqueous acidic media has been explicitly calculated. Molecular geometries of Q and QH2 have been optimized. Protonation of Q, i.e., the formation of QH+ and QH, have been considered. Molecular geometries of these species have been thoroughly optimized. The energy of complexation of these molecules with water have been calculated by optimizing the structures of the hydrated complexes Q · 6H2O, QH2 · 6H2O, QH+ · 6H2O. and QH · 6H2O. The ion–solvent interaction energy of QH+ · 6H2O, in turn, has been calculated by considering the complex QH+ · 6H2O…︁ 2H2O, where the two extra water molecules approach the charge center of the complex QH+ · 6H2O vertically from top and bottom of the quinonoid ring. The standard reduction potential calculated by the CNDO method, 0.8548 V, is somewhat larger than the experimental potential, 0.6998 V, at 25°C. But the INDO value, 0.7085 V, is in excellent agreement with the observed potential. The electrode potential for the plastoquinone (PQ)‐plastohydroquinone (PQH2) couple present in the aqueous pool in chloroplast has been calculated by the INDO method. The basic geometries of PQ, PQH+, and PQH2/sb have been synthesized by adopting the optimized geometries of Q, QH+, and QH2 and considering methyl substituents as well as an isoprenoid side chain containing up to 3 isoprene units with possible geometrical isomerism. The hydrated species PQ · 6H2O, PQH+ · 6H2O, and PQH2 · 6H2O are unstable compared to the isolated species PQ, PQH+, and PQH2, respectively. In fact, we have found that the hydration of PQH+ and PQH2 is much less extensive, and stability arises only when the hydroxyl groups in these two molecules are hydrogen‐bonded to water molecules. But PQH+ is also stabilized through the association of two more water molecules in the vertical direction. For this reason, we have calculated the reduction potential of the PQ/PQH2 system from the energies of the isolated molecules PQH2 and the hydrated species PQH+ · 2H2O. The computed standard reduction potential is 0.2785 V and it yields a potential of 0.07V at pH 7 at 25°C, which is in good agreement with the reduction potential 0.11 V observed for plastoquinone in the aqueous pool in chloroplast. © 1994 John Wiley & Sons, Inc.

The plastoquinone diffusion coefficient in chloroplasts and its mechanistic implications

Pyrene fluorescence quenching by plastoquinone (PQ) was used to estimate lateral diffusion coefficients for PQ-9, decyl PQ and PQ-2 in soybean phosphatidylcholine liposomes and in spinach thylakoid and subthylakoid membranes. All three PQs have diffusion coefficients in the range of (0.1–3)·10 −9 cm 2/s in thylakoids and subthylakoids when measured by the pyrene fluorescence quenching technique, values that are at least two orders of magnitude lower than those measured in phosphatidylcholine liposomes. Our values of PQ diffusion coefficients in thylakoids and subthylakoids are at least one order of magnitude below the minimal values estimated by Mitchell et al. [R. Mitchell et al. (1990) Biophys. J. 58, 1011–1024] if the rate of quinol oxidation by the cytochrome bf complex is determined by quinol binding rather than quinol diffusion. Our results, together with those of Mitchell et al., provide evidence that PQ lateral mobility within the thylakoid membrane determines the rate of quinol oxidation by the cytochrome bf complex. We rationalize our results in terms of a percolation model in which PQ mobility is restricted by thylakoid membrane proteins and conclude that plastocyanin rather than PQ must be responsible for rapid electron transport between PS II and PS I, in agreement with a similar proposal by Lavergne, Joliot and their co-workers [Joliot et al. (1992) Biochim. Biophys. Acta 1101, 1–12; Lavergne et al. (1992) Biochim. Biophys. Acta 1101, 13–22].

The concerted reduction of the high- and low-potential chains of the bf complex by plastoquinol

We have measured the equilibrium constant for the reaction by which the plastoquinol: plastocyanin oxidoreductase ( bf complex) oxidizes plastoquinol. By destroying or removing plastocyanin from spinach thylakoids, the terminal electron acceptor for oxidation of plastoquinol was changed from the oxidized primary donor of Photosystem I (P700 +) to oxidized cytochrome f (cyt f +). Because cytochrome f has a lower mid-point redox potential than P700, the equilibrium constant for oxidation of plastoquinol was lowered by this treatment. Chloroplasts were poised at high ambient redox potential ( E h) (about + 450 mV), to oxidize the cytochromes of the bf complex, and then excited by actinic flashes so that Photosystem II (PS II) produced excess PQH 2. After such treatment, cytochromes b and f became partially reduced following flash excitation. Cytochrome f reduction occurred in two distinct phases: a rapid phase with t 1 2 = about 6–10 ms , and a slow phase with t 1 2 > 100 ms . The rapid reduction phase corresponded well in kinetics and extent to the rapid reduction phase of cytochrome b (cyt b). The slow phase of cyt f reduction was linked to re-oxidation of cyt b, with complex kinetics. From these results, and estimation of the concentration of plastoquinone (PQ) and plastoquinol (PQH 2), we conclude that the concerted reduction of cyt b and f by PQH 2 at the PQH 2 oxidizing site (Q o-site) reached its local thermodynamic equilibrium at the end of the fast phase of cyt f reduction. At this point, there was not sufficient driving force in the PQ/PQH 2 couple to further reduce both cyt f and cyt b, and cyt f reduction could occur only as cyt b became re-oxidized through the PQ-reductase site of the bf complex. From these data, we estimate that, at pH 7.3, the equilibrium constant for the concerted reduction of cyt b and cyt f by the PQ pool was approx. 10, which is consistent with the equilibrium constant calculated from measured values for the midpoint potentials of the redox components. By repeating these experiments at different values of pH, we have shown that the equilibrium constant is pH dependent, and follows the value expected from the measured pH-dependencies of midpoint potentials for the redox components. Our hypothesis is further supported by experiments in which, upon addition of MOA-stilbene, the re-oxidation of cyt b was strongly inhibited and the slow phase of cyt f reduction was greatly slowed, and in which, upon addition of benzoquinone (BQ), the rates of re-oxidation of cyt b as well as the slow phase of cyt f reduction were increased. The BQ-induced effects were largely reversed by addition of MOA-stilbene, indicating that the oxidation of cyt b by BQ is most likely catalyzed by the Q i-site. We conclude that the primary pathway for plastoquinol oxidation at the Q o-site is via a classical oxidant-induced reduction mechanism and does not occur via a semiquinone (SQ) cycle or any other mechanism that allows reduction of the high-potential chain without a concerted reduction of the low-potential chain.