The biomolecule ubiquinone exerts a variety of biological functions

The chemistry of ubiquinone allows reversible addition of single electrons and protons. This unique property is used in nature for aerobic energy gain, for unilateral proton accumulation, for the generation of reactive oxygen species involved in physiological signaling and a variety of pathophysiological events. Since several years ubiquinone is also considered to play a major role in the control of lipid peroxidation, since this lipophilic biomolecule was recognized to recycle alpha-tocopherol radicals back to the chain-breaking form, vitamin E. Ubiquinone is therefore a biomolecule which has increasingly focused the interest of many research groups due to its alternative pro- and antioxidant activity. We have intensively investigated the role of ubiquinone as prooxidant in mitochondria and will present experimental evidences on conditions required for this function, we will also show that lysosomal ubiquinone has a double function as proton translocator and radical source under certain metabolic conditions. Furthermore, we have addressed the antioxidant role of ubiquinone and found that the efficiency of this activity is widely dependent on the type of biomembrane where ubiquinone exerts its chain-breaking activity.     

Modeling the electron transport chain of purple non‐sulfur bacteria

Purple non‐sulfur bacteria (Rhodospirillaceae) have been extensively employed for studying principles of photosynthetic and respiratory electron transport phosphorylation and for investigating the regulation of gene expression in response to redox signals. Here, we use mathematical modeling to evaluate the steady‐state behavior of the electron transport chain (ETC) in these bacteria under different environmental conditions. Elementary‐modes analysis of a stoichiometric ETC model reveals nine operational modes. Most of them represent well‐known functional states, however, two modes constitute reverse electron flow under respiratory conditions, which has been barely considered so far. We further present and analyze a kinetic model of the ETC in which rate laws of electron transfer steps are based on redox potential differences. Our model reproduces well‐known phenomena of respiratory and photosynthetic operation of the ETC and also provides non‐intuitive predictions. As one key result, model simulations demonstrate a stronger reduction of ubiquinone when switching from high‐light to low‐light conditions. This result is parameter insensitive and supports the hypothesis that the redox state of ubiquinone is a suitable signal for controlling photosynthetic gene expression.     

LIGHT-INDUCED REACTIONS OF UBIQUINONE IN PHOTOSYNTHETIC BACTERIUM, CHROMATIUM D

Light-induced changes in ultraviolet absorption were investigatedin a photosynthetic bacterium, Chromatium strain D. The difference spectra (light-minus-dark) of the changes insteady state level of absorption under various experimentalconditions have common maxima (peaks or troughs) at 275 mµ,315 mµ and 340 mµ; The sign of change, however,varied according to the conditions of illumination (see below).From the shape of the difference spectra and for other reasonsdiscussed in the present paper, the changes at 275 mµwere ascribed to the oxidation-reduction changes of ubiquinone. Illumination under aerobic condition caused a reduction of ubiquinonsamounting to about 6–7 percent of total ubiquinone inthe cell. The addition of Na₂S₂O₃ enhanced the amplitude ofthe photoinduced change to about 25% of the total ubiquinonein the cell. The sign of the photoinduced absorption change at 275 mµwas reverted by the presence of malate or succinate as substrate.On illumination under anaerobic condition, there was a photoinducedoxidation of ubiquinone amounting to about 50% of the total.Under aerobic condition the amount was about 6–7 percentof the total. Transitional changes of ubiquinone in the bacterial cell werealso investigated under various experimental conditions. (Received July 24, 1967; )     

Mutations that modify or exclude binding of the QA ubiquinone and carotenoid in the reaction center from Rhodobacter sphaeroides

Three single-site mutations have been introduced at positions close to the Q_A ubiquinone in the reaction centre from Rhodobacter sphaeroides. Two of these mutations, Ala M260 to Trp and Ala M248 to Trp, result in a reaction centre that does not support photosynthetic growth of the bacterium, and in which electron transfer to the Q_A ubiquinone is abolished. In the reaction centre with an Ala to Trp mutation at the M260 residue, electron transfer from the primary donor to the acceptor bacteriopheophytin is not affected by the mutation, but electron transfer from the acceptor bacteriopheophytin to Q_A is not observed. The most likely basis for these effects is that the mutation produces a structural change that excludes binding of the Q_A ubiquinone. A third mutation, Leu M215 to Trp, produces a reaction centre that has an impaired capacity for supporting photosynthetic growth. The mutation changes the nature of ubiquinone binding at the Q_A site, and renders the site sensitive to quinone site inhibitors such as o- phenanthroline. Adopting a similar approach, in which a small residue located close to a cofactor is changed to a more bulky residue, we show that the reaction centre can be rendered carotenoid-less by the mutation Gly M71 to Leu.     

The regulation of the biosynthesis of ubiquinone in the rat.

The urinary excretion of p-hydroxybenzoate was not altered by ubiquinone feeding, but, although decreased considerably, was not eliminated in protein deficiency. The incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone in vivo increased in cold-exposed and p-chlorophenoxyisobutyrate (clofibrate)-fed rats, and these changes were parallel with the changes in the incorporation of [2-14C]mevalonate under these conditions. Starvation, cholesterol feeding and cholic acid feeding resulted in the decreased incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, confirming the decreased ubiquinone synthesis. Feeding exogenous ubiquinone increased the hepatic ubiquinone concentration, but did not cause any decrease in the incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, indicating the absence of a feedback control.     

Oxido-Reduction States and Natural Homologue of Ubiquinone (Coenzyme Q) in Submitochondrial Particles From Etiolated Mung Bean (Phaseolus aureus) Seedlings

A procedure for the isolation of submitochondrial particles in quantity from etiolated Mung bean (Phaseolus aureus) seedlings is described. Using a combination of acetone extraction and 2 systems of thin layer chromatography ubiquinone has been isolated. The isolated ubiquinone migrates coincident with authentic ubiquinone-10 in reversed phase thin layer partition chromatography, gives a positive Craven's test, and has oxidized and reduced spectra characteristic of ubiquinone. The quinone is partially reduced under steady-state electron transfer conditions with both succinate and NADH as substrates and is almost completely reduced under anaerobic conditions with either substrate. The concentration of ubiquinone in the particle is of the order of 4.4 mμmoles per mg particle protein, approximately equal to that found in similar submitochondrial particles from beef heart. It is tentatively concluded that ubiquinone-10 is a functional member of the mitochondrial electron transfer chain of Phaseolus aureus.     

Acquisition of photosynthetic capacity by a reaction centre that lacks the QA ubiquinone; possible insights into the evolution of reaction centres?

A photosynthetically impaired strain of Rhodobacter sphaeroides containing reaction centres with an alanine to tryptophan mutation at residue 260 of the M-polypeptide (AM260W) was incubated under photosynthetic growth conditions. This incubation produced photosynthetically competent strains containing suppressor mutations that changed residue M260 to glycine or cysteine. Spectroscopic analysis demonstrated that the loss of the Q_A ubiquinone seen in the original AM260W mutant was reversed in the suppressor mutants. In the mutant where Trp M260 was replaced by Cys, the rate of reduction of the Q_A ubiquinone by the adjacent (H_A) bacteriopheophytin was reduced by three-fold. The findings of the experiment are discussed in light of the X-ray crystal structures of the wild-type and AM260W reaction centres, and the possible implications for the evolution of reaction centres as bioenergetic complexes are considered.     

The alternative oxidase in roots of poa annua after transfer from high-light to low-light conditions

The activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high-light to low-light conditions, and at the same time from long-day to short-day conditions for four days. As a result of the low-light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O-isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low-light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.     

Metabolism of ubiquinone in relation to thyroxine status

1. Under conditions of thyrotoxicosis induced by feeding rats with iodinated casein, ubiquinone concentration was found to increase in the liver by increased synthesis and by partly decreased catabolism leading to its accumulation. The increased ubiquinone was found primarily in the mitochondrial and supernatant fractions. 2. Supplementing the diet with thyroxine, at less than toxic doses, also increased the synthesis and the concentration of ubiquinone in the liver. 3. In the condition of hypothyroidism obtained by feeding rats with thiouracil the concentration and the synthesis of ubiquinone in the liver showed a small decrease. 4. Synthesis of ubiquinone in liver slices was partially inhibited by addition of thyroxine in vitro. Therefore the activation effect on ubiquinone synthesis of excess of thyroxine in the intact animals appears to be by an indirect mechanism.     

Distinct responses of the mitochondrial respiratory chain to long- and short-term high-light environments in Arabidopsis thaliana

In order to ensure the cooperative function with the photosynthetic system, the mitochondrial respiratory chain needs to flexibly acclimate to a fluctuating light environment. The non-phosphorylating alternative oxidase (AOX) is a notable respiratory component that may support a cellular redox homeostasis under high-light (HL) conditions. Here we report the distinct acclimatory manner of the respiratory chain to long- and short-term HL conditions and the crucial function of AOX in Arabidopsis thaliana leaves. Plants grown under HL conditions (HL plants) possessed a larger ubiquinone (UQ) pool and a higher amount of cytochrome c oxidase than plants grown under low light conditions (LL plants). These responses in HL plants may be functional for efficient ATP production and sustain the fast plant growth. When LL plants were exposed to short-term HL stress (sHL), the UQ reduction level was transiently elevated. In the wild-type plant, the UQ pool was re-oxidized concomitantly with an up-regulation of AOX. On the other hand, the UQ reduction level of the AOX-deficient aox1a mutant remained high. Furthermore, the plastoquinone pool was also more reduced in the aox1a mutant under such conditions. These results suggest that AOX plays an important role in rapid acclimation of the respiratory chain to sHL, which may support efficient photosynthetic performance.     

Kinetic model of the operation of a 2-electron switch in the photosynthetic reaction center of bacteria

A mathematical model, describing the binary oscillation of the concentration of semiquinone form of the secondary acceptor (ubiquinone) in photosynthetic reaction center of purple bacteria is proposed. This model takes into account both the changes of the ubiquinone state when the chromatophores are subjected to short flashes of light, and the successive dark relaxation of the semiquinone form. The model allows to calculate such characteristics as the dependence of the flash number, the stationary level of semiquinone form which is being established, when the flash number increases, the velocity which the concentration of semiquinone form is aspirating towards this stationary level and other characteristics. The model shows that the quantum yield of primary charge separation on the reaction center is higher after odd-number flashes then after even-number flashes.     

The reaction center-LH1 antenna complex of Rhodobacter sphaeroides contains one PufX molecule which is involved in dimerization of this complex.

The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides wild-type cells. PufX is associated with the reaction center-light harvesting 1 (RC-LH1) core complex and plays a key role in lateral ubiquinone/ubiquinol transfer. We have determined the PufX/RC stoichiometry by quantitative Western blot analysis and RC photobleaching. Independent of copy number effects and growth conditions, one PufX molecule per RC was observed in native membranes as well as in detergent-solubilized RC-LH1 complexes which had been purified over sucrose gradients. Surprisingly, two gradient bands with significantly different sedimentation coefficients were found to have a similar subunit composition, as judged by absorption spectroscopy and protein gel electrophoresis. Gel filtration chromatography and electron microscopy revealed that these membrane complexes represent a monomeric and a dimeric form of the RC-LH1 complex. Since PufX is strictly required for the isolation of dimeric core complexes, we suggest that PufX has a central structural role in forming dimeric RC-LH1 complexes, thus allowing efficient ubiquinone/ubiquinol exchange through the LH1 ring surrounding the RC.     

Light-induced reactions of ubiquinone in photosynthetic bacterium, Chromatium D III. Oxidation-reduction state of ubiquinone in intact cells of Chromatium D.

Oxidation-reduction levels of ubiquinone in intact cells ofChromatium under various experimental conditions were measuredby extracting the ubiquinone with organic solvents. Under aerobicconditions, 80% and 75% of the total ubiquinone in Chromatiumexisted in the oxidized form in the dark and in the light, respectively.On illumination in the presence of thiosulfate under aerobicconditions, about 15% of the total ubiquinone was photoreduced.In the presence of KGN under aerobic conditions, about 50% and60% of the total ubiquinone existed in the oxidized form inthe dark and in the light, respectively. Under anaerobic conditions, about 25% and 75% of the total ubiquinoneexisted in the oxidized form in the dark and in the light, respectively.This light-induced oxidation of ubiquinone under anaerobic conditionswas inhibited by piericidin A. These findings are in accordancewith previous findings from spectroscopic experiments. (Received November 11, 1968; )     

Effect of inhibitors on the ubiquinone binding capacity of the primary energy conversion site in the Rhodobacter capsulatus cytochrome bc(1) complex.

A key issue concerning the primary conversion (Q(O)) site function in the cytochrome bc(1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q(O) site is able to accommodate two ubiquinone molecules, the double occupancy model [Ding, H., Robertson, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158]. In the recently reported crystal structures of the cytochrome bc(1) complex, no electron density was identified in the Q(O) site that could be ascribed to ubiquinone. To provide further insight into this issue, we have manipulated the cytochrome bc(1) complex Q(O) site occupancy in photosynthetic membranes from Rhodobacter capsulatus by using inhibitor titrations and ubiquinone extraction to modulate the amount of ubiquinone bound in the site. The nature of the Q(O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to modulation of Q(O) site occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q(O) site inhibitors of the cytochrome bc(1) complex. Addition of stoichiometric concentrations of MOA-stilbene or excess DPA to cytochrome bc(1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g(x)() resonance broadens and shifts from 1. 800 to 1.783. This is exactly the same signal as that obtained when there is only one ubiquinone present in the Q(O) site. Furthermore, addition of MOA-stilbene or DPA to the cytochrome bc(1) complex depleted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line shapes, which remain indicative of one ubiquinone or zero ubiquinones in the Q(O) site, with broad g(x)() resonances at 1. 783 or 1.765, respectively. The results are quite consistent with the Q(O) site double occupancy model, in which MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q(O) site ubiquinones.     

Alternative hydroxylases for the aerobic and anaerobic biosynthesis of ubiquinone in Escherichia coli.

The synthesis of ubiquinone under anaerobic conditions was examined in a variety of strains of Escherichia coli K12. All were shown to synthesize appreciable quantities of ubiquinone 8 when grown anaerobically on glycerol in the presence of fumarate. Under these conditions, ubiquinone 8 was in most cases the principal quinone formed, and levels in the range 50--70% of those obtained aerobically were observed. Studies with mutants blocked in the various reactions of the aerobic pathway for ubiquinone 8 synthesis established that under anaerobic conditions three alternative hydroxylation reactions not involving molecular oxygen are used to derive the C-4, -5, and -6 oxygens of ubiquinone 8. Thus, mutants blocked in either of the three hydroxylation reactions of the aerobic pathway (ubiB, ubiH, or ubiF) are each able to synthesize ubiquinone 8 anaerobically, whereas mutants lacking the octaprenyltransferase (ubiA), carboxy-lyase (ubiD), or methyltransferases (ubiE or ubiG) of the aerobic pathway remain blocked anaerobically. The demonstration that E. coli possesses a special mechanism for the anaerobic biosynthesis of ubiquinone suggests that this quinone may play an important role in anaerobic metabolism.     

Nature, intracellular distribution and formation of terpenoid quinones in Euglena gracilis

1. Light-grown cells of Euglena gracilis strain Z, var. bacillaris and 1224/5g contain phylloquinone, plastoquinone, alpha-tocopherol, alpha-tocopherolquinone and ubiquinone-9 (i.e. ubiquinone with 9 isoprene units/mol.). 2. The concentration (per g. dry wt.) of plastoquinone (and chlorophyll) in light-grown cells of strain Z was governed by the composition of the culture medium and age of the cells. Highest yields of plastoquinone were obtained under autotrophic conditions, the concentration reaching a maximum after 6-8 days' growth. The concentrations were less in heterotrophic media. The concentration of ubiquinone was relatively unaffected by the age of the cells or composition of the medium. 3. In light-grown cells of strain Z plastoquinone, alpha-tocopherolquinone and alpha-tocopherol were mainly localized in the chloroplast; ubiquinone was found to be in the mitochondria. 4. Etiolated (dark-grown) cells of strain Z contained no phylloquinone, plastoquinone or alpha-tocopherolquinone; alpha-tocopherol was present in lower concentrations compared with light-grown cells; ubiquinone concentrations were similar to those for light-grown cells. The presence of alpha-tocopherol in etiolated cells suggested that this chromanol was not entirely confined to the chloroplast. 5. On illumination of etiolated cells of strain Z the chloroplastidic components plastoquinone, alpha-tocopherolquinone and alpha-tocopherol were synthesized in step with chloroplast formation. Ubiquinone concentrations, as expected, were unaffected. 6. [2-(14)C]Mevalonic acid, the specific distal terpenoid precursor, was not incorporated into any of the terpenoid components examined. This was attributed to the impermeability of the cell wall to this compound, rather than to a novel pathway of terpenoid biosynthesis.     

田间小麦叶片光合效率日变化与光合“午睡”的关系

小麦灌浆初期叶片(旗叶)晴天中午光合速率下降(“午睡”)伴随了气孔导度、胞间CO_2浓度下降,而气孔限制值中午升高,进一步证实气孑L中午关闭是光合“午睡”的一个重要原因。叶片光合效率的中午下降并非都伴随着光合“午睡”现象。当两者同时发生时,胞间CO_2浓度降低,而光合速率与气孔导度、胞间CO_2浓度之间的相关性高于光合速率与光合效率之间的相关性。这些事实表明。即使光合效率中午下降是光合“午睡”的部分原因,但较之气孔中午关闭只是一个次要原因。     

Transfer of light-induced electron-spin polarization from the intermediary acceptor to the prereduced primary acceptor in the reaction center of photosynthetic bacteria.

In reaction centers and chromatophores of photosynthetic bacteria strong light-induced emissive ESR signals have been found, not only after a flash but also under continuous illumination. The signal, with g = 2.0048 and delta Hpp = 7.6 G, is only present under reducing conditions in material in which the primary acceptor, ubiquinone, U and its associated high-spin ferrous ion are magnetically uncoupled. its amplitude under continuous illumination is strongly dependent on light intensity and on microwave power. The emissive signal is attributed to the prereduced primary acceptor, U-, which becomes polarized through transfer of spin polarization by a magnetic exchange interaction with the photoreduced, spin polarized intermediary acceptor, I-. A kinetic model is presented which explains the observed dependence of emissivity on light intensity and microwave power. Applying this analysis to the light saturation data, a value of the exchange rate between I- and U- of 4.10(8) s-1 is derived, corresponding to an exchange interaction of 3--5 G.     

Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.

The biosynthetic origin of the oxygen atoms of ubiquinone 8 from aerobically grown Escherichia coli was studied by 18O labeling. An apparatus was developed which allowed the growth of cells under a defined atmosphere. Mass spectral analysis of ubiquinone 8 from cells grown under highly enriched 18O2 showed that three oxygen atoms of the quinone are derived from molecular oxygen. It was established that the molecular oxygen is incorporated into the two methoxyl groups (at C-5 and C-6) and one of the carbonyl positions of the ubiquinone molecule by demonstrating that only one of the incorporated oxygens will exchange with water under acidic conditions that specifically catalyze the exchange of carbonyl, but not methoxyl, oxygens. That the C-4 carbonyl oxygen is derived from molecular oxygen was shown by the incorporation of three atoms of 18O2 into ubiquinone 8 biosynthesized from added 4-hydroxybenzoic acid. Comparison of ubiquinone 8 and menaquinone 8 from E. coli grown under 18O2 confirmed that the labeled carbonyl oxygen of the [18O2]ubiquinone 8 is incorporated biosynthetically and not by chemical exchange in the cell. It is concluded that the three hydroxylation reactions involved in the pathway for the aerobic biosynthesis of ubiquinone are all catalyzed by monooxygenases. The implications of this study for the anaerobic biosynthesis of ubiquinone 8 in E coli are discussed.