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Physicochemical properties of charge-transfer complexes of plastoquinone and α-tocopherol quinone, and their possible role in vivo
Abstract
It has been found that the solubility of PQH2 and α-TQH2 in hexane increased with the increase in PQ and α-TQ concentrations, respectively, that is connected with the formation of quinhydrone-type charge-transfer complexes. Measurements of the solubility of both prenylquinones and their reduced forms in hexane and acetone, at − 30°C and room temperature, showed a much higher affinity of the quinol forms for acetone than for hexane. In the case of quinones, the difference in affinity was not significant. The possibility of charge-transfer complex formation by PQ and α-TQ in thylakoid membranes and the influence of such complexes on the diffusion of PQH2 and α-TQH2 molecules have been considered.
... Nevertheless, it must cross the membrane to reach the oxidation site at the cytochrome b 6 f complex. Based on the results of experiment performed in several model systems214215216217, it was suggested that the oxidized and reduced PQ molecules form a charge–transfer complex that facilitates penetration of hydrophobic membrane interior by PQH 2 molecules. It was shown in these studies that the formation of the charge–transfer complex increases PQH 2 solubility in hydrophobic solvents, as well as decreases the content of PQH 2 at the interphase region of monogalactosyldiacylglycerol monolayer [215,216]. ...
... Based on the results of experiment performed in several model systems214215216217, it was suggested that the oxidized and reduced PQ molecules form a charge–transfer complex that facilitates penetration of hydrophobic membrane interior by PQH 2 molecules. It was shown in these studies that the formation of the charge–transfer complex increases PQH 2 solubility in hydrophobic solvents, as well as decreases the content of PQH 2 at the interphase region of monogalactosyldiacylglycerol monolayer [215,216]. A tendency to occupy different membrane regions by the oxidized and reduced forms of PQ was found in anisotropy studies of diphenyl- hexatriene [218], the fluorescent probe that monitors membrane lipid order. ...
Isoprenoid quinones are one of the most important groups of compounds occurring in membranes of living organisms. These compounds are composed of a hydrophilic head group and an apolar isoprenoid side chain, giving the molecules a lipid-soluble character. Isoprenoid quinones function mainly as electron and proton carriers in photosynthetic and respiratory electron transport chains and these compounds show also additional functions, such as antioxidant function. Most of naturally occurring isoprenoid quinones belong to naphthoquinones or evolutionary younger benzoquinones. Among benzoquinones, the most widespread and important are ubiquinones and plastoquinones. Menaquinones, belonging to naphthoquinones, function in respiratory and photosynthetic electron transport chains of bacteria. Phylloquinone K(1), a phytyl naphthoquinone, functions in the photosynthetic electron transport in photosystem I. Ubiquinones participate in respiratory chains of eukaryotic mitochondria and some bacteria. Plastoquinones are components of photosynthetic electron transport chains of cyanobacteria and plant chloroplasts. Biosynthetic pathway of isoprenoid quinones has been described, as well as their additional, recently recognized, diverse functions in bacterial, plant and animal metabolism.
... The absorption spectra of each individual peaks were collected from the PDA equipped in the liquid chromatography system. To estimate the stoichiometry of α-TQ per PSII-TPP complex, β-carotene is chosen as a relatively specific internal reference for estimating the amount of PSII as both α-TQ and β-carotene are highly soluble in n-hexane 94,95 and accessible to the extracting reagents. The maximum absorption wavelength of α-TQ (268 nm) was used to monitor the compounds eluted from the HPLC column. ...
Photosystem II (PSII) catalyzes water oxidation and plastoquinone reduction by utilizing light energy. It is highly susceptible to photodamage under high-light conditions and the damaged PSII needs to be restored through a process known as the PSII repair cycle. The detailed molecular mechanism underlying the PSII repair process remains mostly elusive. Here, we report biochemical and structural features of a PSII-repair intermediate complex, likely arrested at an early stage of the PSII repair process in the green alga Chlamydomonas reinhardtii. The complex contains three protein factors associated with a damaged PSII core, namely Thylakoid Enriched Factor 14 (TEF14), Photosystem II Repair Factor 1 (PRF1), and Photosystem II Repair Factor 2 (PRF2). TEF14, PRF1 and PRF2 may facilitate the release of the manganese-stabilizing protein PsbO, disassembly of peripheral light-harvesting complexes from PSII and blockage of the QB site, respectively. Moreover, an α-tocopherol quinone molecule is located adjacent to the heme group of cytochrome b559, potentially fulfilling a photoprotective role by preventing the generation of reactive oxygen species.
... Recently, PQH 2 -9 has been suggested to act as a singlet oxygen scavenger in photosystem II, similarly to α-Toc [36], however its singlet oxygen scavenging properties have not been investigated until now. Although its quenching rate constant is three to six times less that that of α-Toc in polar and hydrophobic solvents, respectively, it is known that the ring of α-Toc is located at the membrane surface [55], while PQH 2 -9 is located within the hydrophobic membrane interior [41,[55][56][57] which makes possible its efficient singlet oxygen scavenging within the membrane in contrast to α-Toc. ...
Singlet oxygen quenching rate constants for tocopherol and tocotrienol homologues have been determined in organic solvents of different polarities, as well as for other biological prenyllipids such as plastoquinol, ubiquinol, and alpha-tocopherolquinol. The obtained results showed that the quenching activity of tocochromanols was mainly due to the chromanol ring of the molecule and the activity increased with the number of the methyl groups in the ring and solvent polarity. Among prenylquinols, alpha-tocopherolquinol was the most active scavenger of singlet oxygen followed by ubiquinol and plastoquinol. The oxidation products of tocopherols were identified as 8a-hydroperoxy-tocopherones which are converted to the corresponding tocopherolquinones under acidic conditions. The primary oxidation products of prenylquinols, containing unsaturated side chains, were the corresponding prenylquinones that were further oxidized to hydroxyl side-chain derivatives. In the case of plastochromanol, the gamma-tocotrienol homologue found in some seed oils, mainly the hydroxyl derivatives were formed, although 8a-hydroperoxy-gamma-tocopherones were also formed to a minor extent, both from plastochromanol and from its hydroxyl, side-chain derivatives. The obtained results were discussed in terms of the activity of different prenyllipids as singlet oxygen scavengers in vivo.
Plastoquinone-9 (PQ-9) is an essential component of photosynthesis that carries electrons in the linear and alternative electron transport chains, and is also a redox sensor that regulates state transitions and gene expression. However, a large fraction of the PQ pool is located outside the thylakoid membranes, in the plastoglobules and the chloroplast envelopes, reflecting a wider range of functions beyond electron transport. This review describes new functions of PQ in photoprotection, as a potent antioxidant, and in chloroplast metabolism as a cofactor in the biosynthesis of chloroplast metabolites. It also focuses on the essential need for tight environmental control of PQ biosynthesis and for active exchange of this compound between the thylakoid membranes and the plastoglobules. Through its multiple functions, PQ connects photosynthesis with metabolism, light acclimation, and stress tolerance.
In the present study, we have demonstrated that membrane-free extracts of etiolated shoots of Phaseolus coccineus seedlings show tocopherol oxidase activity. For this reaction, presence of membrane lipids, such as lecithin and mixture of plant lipids was required. The rate of the reaction was the highest for α-tocopherol and decreased in the order α ≫ β > γ > δ tocopherols. In the case of α-tocopherol, the main oxidation product was α-tocopherolquinone, while for the other tocopherol homologues the dominant products were other derivatives. When the enzyme activity was measured in leaves, hypocotyls and roots of etiolated seedlings of P. coccineus, the oxidase activity was the highest in extracts of leaves and decreased towards the roots where no activity was detected. The effect of hydrogen peroxide and of different inhibitors on the reaction suggest that tocopherol oxidase does not belong to peroxidases or flavin oxidases but rather to multi-copper oxidases, such as polyphenol oxidases or laccases. On the other hand, catechol, the well-known substrate of polyphenol oxidases and laccases, was not oxidized by the enzyme, indicating a high substrate specificity of the tocopherol oxidase.
It has been found that plastoquinone (PQ) and α-tocopherol quinone (α-TQ) form quinhydrone-type charge-transfer (CT) complexes on PQH2 and α-TQH2, respectively, in oleic, linoleic and linolenic acid throughout the whole range of temperatures examined, i.e., from −30°C to 60°C. The CT spectra of the PQ-PQH2 complex at room temperature showed one peak at 371.8–383 nm and a shoulder at 454–494 nm, depending on the fatty acid used. The corresponding CT spectra for the α-TQ—α-TQH2 complex were similar in shape with one peak at 371.4–378.1 nm and a shoulder at 439–469 nm. The temperature dependences of CT spectra were not large above the melting point of fatty acids. On the other hand, the changes in CT spectra in the frozen state were striking. A long-wavelength shift of both CT bands (up to 90 nm) and an increase in intensities of the bands, especially of the long-wavelength band, was observed, and as a result the intensity ratio of both bands was reversed with the decrease in temperature. The observed effects were explained in terms of different orientational isomers of the complex, the relative quantities of which varied with temperature changes.
It has been found that plastoquinone-9 and α-tocopherol quinone form charge-transfer complexes with their corresponding reduced forms in anhydrous dipalmitoyl phosphatidylcholine and octadecane throughout the whole temperature range examined, i.e. from -30 to 60°C. The charge-transfer spectra of both prenylquinones in lecithin showed the presence of only one band with λmax in the range 520–600 nm, depending on temperature. There was a gradual long-wavelength shift of λmax of the band observed with decrease in temperature. The intensity of the band was quite constant. Only at the lowest temperatures was a sudden intensity decrease, caused by precipitation of the hydroquinone form, observed. On the other hand, the charge-transfer spectra of both prenylquinones in octadecane showed the presence of two bands with strong temperature dependence. On freezing, the intensity of these two bands increased with a simultaneous long-wavelength shift of both maxima. The results are discussed in terms of different molecular structures of the complex in both systems.
We have found that in isolated spinach thylakoids, plastoquinone-pool (PQ-pool), after its photoreduction, undergoes dark-reoxidation with the half-time of 1/2 = 43 3 s. To explain the observed rates of PQ-pool reoxidation, a nonenzymatic plastoquinol (PQH2) autoxidation under molecular oxygen and an enzymatic oxidation by the low-potential form of cytochrome b-559 (cyt. b-559LP), as the postulated PQ-oxidase in chlororespiration, were investigated. It was found that the autoxidation rate of PQH2 in organic solvents and liposomes was too low to account for the observed oxidation rate of PQH2 in thylakoids. The rate of cyt. b-559LP autoxidation in isolated Photosystem II was found to be similar (1/2 = 26 5 s) to that of the PQ-pool. This suggests that the LP form of cyt. b-559 is probably responsible for the PQ-oxidase activity observed during chlororespiration.
The surface pressure-area isotherms of pure plastoquinone-9 (PQ-9), plastoquinone-3 (PQ-3), alpha-tocopherol quinone (alpha-TQ), their reduced (hydroquinone) forms and mixtures of these molecules with monogalactosyldiacylglycerol (MGDG) have been studied by a monolayer technique. The collapse pressures of all hydroquinones (QH2) were higher than those of the corresponding quinones (Q), the difference being highest between PQ-9 and PQH2-9. The limiting molecular areas of hydroquinones were higher than those of the corresponding quinones except for alpha-TQH2. All Q-QH2 mixtures showed miscibility throughout the whole range of the components' ratios. There was no deviation from the additivity rule observed for any of the Q-QH2 mixture, as well as for the mixtures of MGDG with PQ-3, PQH2-9, alpha-TQ and alpha-TQH2. On the other hand, PQ-9/MGDG and PQH2-3/MGDG mixtures showed positive and negative deviations, respectively. All the isotherms of Q-MGDG and QH2-MGDG mixtures showed a kink point above the collapse pressure of the Q or QH2 examined, indicating that with the increase in surface pressure, Q or QH2 were gradually squeezed out from the monolayer. The percent content of Q and QH2 in the monolayer as a function of surface pressure was also calculated. The hydroquinones were more difficult to remove from monolayers than the corresponding quinones, and among the investigated quinones, PQ-9 was most easily and alpha-TQ most difficulty squeezed out. The surface pressure-area isotherms of the three-component mixtures of PQ-9/PQH2-9/MGDG showed a shift to lower molecular areas in comparison with the corresponding two-component mixtures, especially at higher surface pressures. This indicates that the presence of PQ-9 lowered the PQH2-9 content in the monolayer, especially at higher pressures, which was explained by charge-transfer complex formation upon interaction of PQ-9 with PQH2-9. The comparison of surface potential-area isotherms of PQ-9/PQH2-9/MGDG mixtures with those of the corresponding binary mixtures also suggest charge-transfer interaction between PQ-9 and PQH2-9. The orientation and localization of the investigated quinones and quinols in the thylakoid membrane and significance of charge-transfer interactions in functioning of PQ-9 has been discussed.
It was found that plastoquinol-9, ubiquinol-10 and alpha-tocopherol quinol show intrinsic fluorescence in organic solvents and in liposomes. Their fluorescence spectra in solution showed the presence of one emission band with maximum intensity in the range 319.0-327.0 nm for plastoquinol and 321.5-326.5 nm for alpha-tocopherol quinol, which is the longest wavelength shifted in polar solvents. The emission band at about 371 nm for ubiquinol was not sensitive to solvent polarity. For all three prenylquinones the fluorescence quantum efficiency changed significantly in solvents of different polarities, being the highest in ethanol and the lowest in hexane in the case of plastoquinol and alpha-tocopherol quinol, whereas ubiquinol fluorescence showed the opposite effect. These spectral parameters were applied to determination of prenylquinol localization in liposome membranes.
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 alpha-tocopherol or alpha-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.
We have examined scavenging of a superoxide by various prenyllipids occurring in thylakoid membranes, such as plastoquinone-9, alpha-tocopherolquinone, their reduced forms, and alpha-tocopherol, measuring oxygen uptake in hexane-extracted and untreated spinach thylakoids with a fast oxygen electrode under flash-light illumination. The obtained results demonstrated that all the investigated prenyllipids showed the superoxide scavenging properties, and plastoquinol-9 was the most active in this respect. Plastoquinol-9 formed in thylakoids as a result of enzymatic reduction of plastoquinone-9 by ferredoxin-plastoquinone reductase was even more active than the externally added plastoquinol-9 in the investigated reaction. Scavenging of superoxide by plastoquinol-9 and other prenyllipids could be important for protecting membrane components against the toxic action of superoxide. Moreover, our results indicate that vitamin K(1) is probably the most active redox component of photosystem I in the generation of superoxide within thylakoid membranes.
A rapid, sensitive fluorescence method was applied here for detection of oxidized tocopherol quinones in total plant tissue extracts using HPLC, employing a post-column reduction of these compounds by a Zn column. Using this method, we were able to detect both alpha- and gamma-tocopherol quinones in Chlamydomonas reinhardii with a very high degree of sensitivity. The levels of both compounds increased under high light stress in the presence of pyrazolate in parallel to a decrease in the content of the corresponding tocopherols. The formation of tocopherol quinones from tocopherols was apparently due to their oxidation by singlet oxygen, which is formed in photosystem II under high light stress. alpha-Tocopherol quinone was also detected in a variety of higher plants of different age, and its level was found to increase during senescence in leaves grown under natural conditions. In contrast to alpha-tocopherol quinone, gamma-tocopherol quinone was not found in the higher plant species investigated with the exception of young runner bean leaves, where the levels of both compounds increased dramatically during cold and light stress. Taking advantage of native fluorescence of the reduced alpha-tocopherol quinone (alpha-tocopherol quinol), it can be detected in plant tissue extracts with a high sensitivity. In young runner bean leaves, alpha-tocopherol quinol was found at a level similar to alpha-tocopherol.
Figure 1 shows the zigzag scheme of the molecular machinery of photosynthesis with the vectorial pathways of electrons, protons and hydrogens derived from pulse spectroscopic studies (1,2). The time sequence of the reaction patterns has been evaluated as follows. (1) Excitation of Chl-aI and Chl-aII. (2) Vectorial ejection of electrons from Chl-a
I* and Chl-a
II* at the membrane inside to the outside and electric field generation. (3) Oxidation of H2O, reduction of NADP+, reduction of PQ, and reoxidation. (4) Proton translocation into the inner phase through protolytic reactions with the charges at the outer and inner surface of the membrane. (5) Discharging of the energized state through efflux of protons. (6) Formation of free ATP from ADP + P by the energy released with the efflux of H+ via the ATP-synthetase. In toto the H+-translocation corresponds to a H+-circulation. This cycle is closed, however, only if between system I and II a hydrogen (H+ plus e) is translocated from the outside to the inside (Fig. 1). Based on the protolytic properties of redox reactions in quinone systems, the PQ pool was inferred to be a candidate for a “pump” for hydrogen (H+ + e) from the membrane outside to the inside (3). Recently it was discussed that a pump not coupled to redox reactions may be responsible for this H-transfer (4).
Surface-active properties of ubiquinones and ubiquinols have been investigated by monomolecular-film techniques. Stable monolayers are formed at an air/water interface by the fully oxidized and reduced forms of the coenzyme; collapse pressures and hence stability of the films tend to increase with decreasing length of the isoprenoid side chain and films of the reduced coenzymes are more stable than those of their oxidized counterparts. Ubiquinone with a side chain of two isoprenoid units does not form stable monolayers at the air/water interface. Mixed monolayers of ubiquinol-10 or ubiquinone-10 with 1,2-dimyristoyl phosphatidylcholine, soya phosphatidylcholine and diphosphatidylglycerol do not exhibit ideal mixing characteristics. At surface pressures less than the collapse pressure of pure ubiquinone-10 monolayers (approx. 12mN.m(-1)) the isoprenoid chain is located substantially within the region occupied by the fatty acyl residues of the phospholipids. With increasing surface pressure the ubiquinones and their fully reduced equivalents are progressively squeezed out from between the phospholipid molecules until, at a pressure of about 35mN.m(-1), the film has surface properties consistent with that of the pure phospholipid monolayer. This suggests that the ubiquinone(ol) forms a separate phase overlying the phospholipid monolayer. The implications of this energetically poised situation, where the quinone(ol) is just able to penetrate the phospholipid film, are considered in terms of the function of ubiquinone(ol) as electron and proton carriers of energy-transducing membranes.
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 . 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.
Using direct chemical measurement, the level of plastoquinone-9 in chloroplast thylakoids of Pisum sativum (pea) was found to be 21 mol per 1000 mol chlorophyll, a value in close agreement with previous estimates made by direct kinetic and optical methods. Mechanical fragmentation of isolated thylakoids ensured removal of a major source of plastoquinone-9 contamination, the plastoglobuli, and provided samples containing predominantly appressed and non-appressed membranes. The level of plastoquinone-9 was 0.6–0.7% of the acyl-lipid matrix (mol/mol) in both thylakoid regions, a result consistent with the role of plastoquinone-9 as a long-range mobile electron/proton carrier.
The role of plastoquinone as a mobile redox carrier linking photosystem II and the cytochrome b6−f complex is considered. It is proposed that plastoquinone is located primarily within the fluid bilayer-midplane region of the thylakoid membrane and thus can move laterally at very fast rates corresponding to a microscopic diffusion coefficient of 10−6 cm2·s−1. Because of the presence of integral proteins the diffusion path will be tortuous and extended, giving rise to a sub-macroscopic diffusion coefficient which is lower than the above value. Even so it is concluded that within the half-time for electron donation to the cytochrome b6−f complex there is adequate time for plastoquinone to diffuse over a distance equivalent to the radius of a granum membrane.
This chapter describes plastoquinones in algae and higher plants. Three new forms of naturally occurring plastoquinone have been identified in Euglena gracilis. They include phytylplastoquinone (2,3-dimethy-5-phytyl-l,4-benzoquinone), phytylplastoquinol monomethyl ether [2,3-dimethyl-4-methoxy-5 (or 6)-phytylphenol], and 1-omethyl-2-demethylphytylplastoquinol. The biochemistry of plastoquinone and other chloroplast quinone has been reviewed extensively by Wallwork and Pennoc, Wallwork and Crane, and Morton. Plastochromanols, cyclized derivatives of PQA, have been described by Peake, Dunphy, and Pennock. An integrated view of how chloroplast quinones fit into a general electron transport scheme is provided by Trebst. Plastoquinones in algae have been studied by Takamiya, Carr, Allen, Oku, Senger and Frickel–Faulstich, and Hanigk and Lichtenthaler. Work with chloroplast-rich particles of sugar beet and Euglena gracilis has demonstrated the biosynthesis of nonaprenyltoluquinol (2-demethylplastoquinol-9, a postulated PQA precursor) and an octaprenyltoluquinol from [U-14C] homogentisic acid and other forms of labeled homogentisic acid in the presence of protein-bound polyprenylpyrophosphates. Cell-free homogenates of Euglena can also carry out the biosynthesis of nonaprenyltoluquinol and an octaprenyltoluquinol.
A simple, rapid, graphical method for the ultraviolet spectrophotometric analysis of two-component systems uses the ratio of observed absorbances at two selected wave lengths, one of which is an "isoabsorptive" point if possible. This permits a straight-line plot of the ratio of the observed absorbances vs. relative composition, as derived from Beer's law. The method may be applied even though no isoabsorptive point exists, by use of a plot of calculated ratio vs. composition. It can be extended to a three-component system if desired. Speed and ease in setting up an analysis are obtained, as knowledge of only three absorptivities is needed for a two-component analysis if an isoabsorptive point is used; these values may be readily obtained from the user's files, the literature, or another laboratory.
The absorption maximum of halorhodopsin in a membrane fraction prepared from the cells of Halobacterium halobium under low-salt conditions shifted to longer wavelenghts upon addition of NaCl (Ogurusu, T., Maeda, A., Sasaki, N. and Yoshizawa, T. (1981) J. Biochem. (Tokyo) 90, 1267–1273). This bathochromic shift was due to chloride, not sodium. Bromide and iodide were also effective. The bathochromic shift of the absorption maximum was not accompanied by any change in the isomer composition of retinal in halorhodopsin. The same ionic species were essential for the formation of the hypsochromic photoproduct at −75°C. These effects of NaCl on halorhodopsin are discussed in terms of the presence of the two forms of halorhodopsin, a form binding chloride and a chloride-free form.
A study of signals, light-induced at 77 K in O2-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q−AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin− signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin− signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo.
Physiological quinones carrying isoprenoid side chains have been compared with homologues lacking the side chain, for their ability to carry electrons and protons from dithionite to ferricyanide, trapped in liposomes. Six differential observations were made: 1.(1) Plastoquinone and ubiquinones, with a side chain of more than two isoprene units, are by far better mediators than their short-chain homologues. Also other benzoquinones lacking a long side chain are poor catalysts, except dimethyl-methylenedioxy-p-benzoquinone, a highly autooxidizable compound. Tocopherol is a good catalyst.2.(2) Vitamin K-1 and K-2 are poor mediators compared to vitamin K-3.3.(3) The reaction catalyzed by quinones carrying long isoprenoid side chains has an about three-fold higher activation energy, irrespective of the catalytic efficiency.4.(4) The reaction catalyzed by quinones lacking a long side chain follows pseudo first-order kinetics, while the reaction with quinones carrying a long side chain is of apparently higher order.5.(5) The rate with ubiquinone-1 is increasing with increasing pH, while with ubiquinone-9 it is decreasing.6.(6) The reaction mediated by short-chain quinones seems to be saturated at lower dithionite concentration.We conclude that isoprenoid quinones are able to translocate electrons and protons in lipid membranes, and that the side chain has a strong impact on the mechanism. This and the relevance of the model reaction for electron and proton transport in photosynthesis and respiration is discussed.
The effect of two ubiquinones of different side chain length (Q-3; Q-9), on the fluidity of phospholipid vesicles has been investigated using stearic acid spin labels. While both oxidized quinones have a disordering effect on the lipid bilayers, the reduced forms behave in an opposite way, in that Q-3 enhances and Q-9 decreases the order of the bilayer. The ordering effect of reduced Q-3 and the attendant decreased motional freedom in the bilayer might be the result of the insertion and stacking of the quinone between the phospholipid molecules in the bilayer. Such insertion might be related to the incapability of short-chain quinones in restoring NADH oxidation in Q-depleted mitochondria.
1. The penetration of alpha-tocopherol and seven of its derivatives, and five compounds in the ubiquinone series, having differing chain lengths, into monolayers at the air/water interface of 11 different synthetic phospholipids and cholesterol was investigated; the properties of mixed monolayers of the tocopherols and of ubiquinones with phospholipids were also studied. 2. Penetration of alpha-tocopherol into diarachidonylglycerylphosphorycholine was approximately constant for molar ratios of tocopherol/phospholipid ranging from 0.4:1.0 to 2.0:1.0. 3. Tocopherols with shorter or longer side chains than alpha-tocopherol had a lesser ability to penetrate monolayers of phospholipid molecules with 16 or more carbon atoms in their acyl chains. 4. All the tocopherols penetrated more readily as unsaturation in the phospholipids was increased, and their penetration into mixed monolayers of phospholipids was greatly facilitated by the presence of relatively small quantities of unsaturated phospholipid molecules. 5. There was relatively little interaction between the tocopherols and cholesterol, or between the ubiquinones and phospholipids. 6. The possible significance of the observed interactions between alpha-tocopherol and polyunsaturated phospholipids is discussed in relation to the biochemical actions of alpha-tocopherol in vivo. 7. It is suggested that fluidity of the lipid bilayer in membranes containing polyunsaturated phospholipids may allow alpha-tocopherol to interact in a dynamic manner with a number of phospholipid molecules.
Ubiquinones (n = 1,2,3,4,7,9,10) and ubiquinols (n = 1,2,3,4,10) were incorporated into ordinary (protonated) or perdeuterated dimyristoyl phosphatidylcholine vesicles and were found to have significant local molecular motion. The motion of the quinone ring, as judged from the linewidth of the OCH3 proton resonances, decreased in longer-chain ubiquinones. Minimum values for the transverse mobility (flip-flop rates) of ubiquinones-1,2,3,4,10, measured with the aid of lanthanide shift reagents, suggest that they are all able to function in a protonmotive 'Q cycle' during electron transport. As the length of the side chain increases beyond 1 isoprenoid unit, the quinone/quinol ring tends to be deeper in the outer monolayer of small sonicated vesicles and in both monolayers of larger freeze-thaw vesicles, but little or no change in depth is observed in the inner monolayer of small vesicles. The ubiquinol rings are closer to the membrane surface than are the ubiquinone rings. For side chain n = 9 or 10, a second resonance from the OCH3 protons of ubiquinones and ubiquinols in vesicles appears in the 2H-NMR spectrum. This is due to the presence of two types of vesicles with different ubiquinone/phospholipid ratios.
It has been found that plastoquinone (PQ) and alpha-tocopherol quinone (alpha-TQ) can form quinhydrone-type charge-transfer complexes on PQH2 and alpha-TQH2, respectively, both in the crystalline state and in solutions of organic solvents. The charge-transfer spectra of PQ/PQH2 mixtures in hydrophobic solvents showed two bands: one at 349-358 nm, the other at 430-440 nm, one charge-transfer band at 351-355 nm occurring in water-miscible solvents. The intensity ratio of these two bands varied with changing PQ/PQH2 ratio. The charge-transfer spectra of alpha-TQ/alpha-TQH2 mixtures in all solvents investigated showed one peak at 361-367 nm and a broad shoulder within the range 400-540 nm, whose shape varied depending on the solvent used. In the infrared spectrum of PQ and alpha-TQ (1700-1600 cm(-1)) splitting of the carbonyl band occurred and was caused by the presence of two peak. In the spectra of quinhydrones the splitting disappeared, this being brought about by the appearance of a new peak at the position of splitting, which originated from the complexed quinones. The possibility of the formation of such complexes in thylakoid membranes is discussed.
- Barr