Synthetic quinones influencing herbicide binding and Photosystem II electron transport. The effects of triazine-resistance on quinone binding properties in thylakoid membranes

We have analyzed the binding of synthetic quinones and herbicides which inhibit electron transport at the acceptor side of Photosystem II (PS II) of the photosynthetic electron-transport chain in thylakoid membranes. These data show that quinones and PS II-directed herbicides compete for binding to a common binding environment within a PS II region which functions as the Q⁻ / PQ oxidoreductase. We observed that (1) synthetic quinones cause a parallel inhibition of electron transport and [¹⁴C]herbicide displacement, and (2) herbicide binding is affected both by the fully oxidized and fully reduced form of a quinone. Quinone function and inhibitor binding were also investigated in thylakoids isolated from triazine-resistant weed biotypes. We conclude the following. (1) The affinity of the secondary accepting quinone, B, is decreased in resistant thylakoids. (2) The observation that the equilibrium concentration of reduced Q after transferring one electron to the acceptor side of PS II is increased in resistant as compared to susceptible chloroplasts may be explained both by a decrease in the affinity of PQ for the herbicide / quinone binding environment, and by a decrease of the midpont redox potential of the B / B⁻ couple. (3) The binding environment regulating quinone and herbicide affinity may be divided roughly into two domains; we suggest that the domain regulating quinone head-group binding is little changed in resistant membranes, whereas the domain-regulating quinone side-group binding (and atrazine) is altered. This results in increased inhibitory activity of tetrachloro-p-benzoquinone and phenolic herbicides, which are hypothesized to utilize the quinone head-group domain. The two domains appear to be spatially overlapping because efficient atrazine displacement by tetrachloro-p-benzoquinone is observed.     

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation.

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain. Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

Analytical precision and repeatability of respiratory quinones for quantitative study of microbial community structure in environmental samples

Microbial community structure is one of the important factors controlling the pollutant-degrading capacity of ecosystems. The analysis of microbial quinones has gained increased recognition as a simple and useful approach for studying microbial structure in environmental samples. The analytical precision of quinone characterization using high performance liquid chromatograph (HPLC) with a UV-detector was studied in this study. Activated sludge was used as a typical mixed culture. The coefficient of variation of quinone content was lower than 6%, and that of microbial diversity calculated from the composition of quinones was as low as 3%. Statistical analyses on the analytical precision of quinones demonstrated that the critical value of dissimilarity between two quinone profiles of activated sludge, which is used to make a judgement whether the two quinone profiles are different or not, is 0.1 for the analytical method used in this study. The values of minimum biomass required for quinone analysis to have a reliable analytical result of microbial quinones were 2 mg-dry-cell for activated sludge.     

Quinone reductase 2 is a catechol quinone reductase

The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.     

Characterization of quinone tautomerase activity in the hemolymph of Sarcophaga bullata larvae

The hemolymph of Sarcophaga bullata larvae was activated with either zymosan or proteolytic enzymes such as chymotrypsin or subtilisin and assayed for phenoloxidase activity by two different assays. While oxygen uptake studies readily attested to the wide specificty of activated phenoloxidase, visible spectral studies failed to confirm the accumulation of quinone products in the case of 4-alkyl substituted catechols such as N-acetyldopamine and N-β-alanyldopamine. Sepharose 6B column chromatography of the activated hemolymph resolved phenoloxidase activity into two fractions, designated as A and B. Peak A possessed typical o-diphenoloxidase (o-diphenol, oxygen oxidoreductase EC 1.10.3.1) activity, while peak B oxidized physiologically important catecholamine derivatives such as N-acetyldopamine, N-acetylnorepinephrine, and N-β-alanyldopamine into N-acetylnorepinephrine, N-acetylarterenone, and N-β-alanylnorepinephrine, respectively, and converted 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol into 3,4-dihydroxymandelic acid, 3,4-dihydroxybenzaldehyde, and 2-hydroxy-3′,4′-dihydroxyacetophenone, respectively. These transformations are consistent with the conversion of phenoloxidase-generated quinones to quinone methides and subsequent non-enzymatic transformations of quinone methides. Accordingly, Peak B contained both o-diphenoloxidase activity and quinone tautomerase activity. Sepharose 6B column chromatography of unactivated hemolymph resulted in the separation of quinone tautomerase from prophenoloxidase. The tautomerase rapidly converted both chemically made and mushroom tyrosinase-generated quinones to quinone methides. Thus the failure to observe the accumulation of quinones with N-acyl derivatives of dopamine and related compounds in the whole hemolymph is due to the rapid conversion of these long lived toxic quinones to short lived quinone methides. The latter, being unstable, undergo rapid non-enzymatic transformations to form side-chain-oxygenated products that are non-toxic. The possible roles of quinone isomerase and its reaction products—quinone methides—as essential components of sclerotization of cuticle and defense reaction of Sarcophaga bullata are discussed.     

Heterologous expression and biochemical characterization of an NAD(P)H:quinone oxidoreductase from the hemiparasitic plant Triphysaria versicolor

Quinones are widespread secondary metabolites that function as signal molecules between organisms in the rhizosphere. Quinones are particularly important in the exchange of chemical signals between plant roots, a phenomenon classically termed allelopathy. The bioactivity of quinones is due in large part to radical intermediates formed during redox cycling between quinone and hydroquinone states. In order to investigate the role of quinone oxidoreductases in processing quinone signals exchanged between plant roots, we characterized an NAD(P)H-dependent quinone reductase expressed in roots of the parasitic plant Triphysaria versicolor (TvQR2). The predicted amino acid sequence encoded by TvQR2 shares homology with quinone reductases from Archaea, Eubacteria and Eukaryota organisms. The complete TvQR2 cDNA was cloned into the fungus Pichia pastoris and the heterologous protein purified. The recombinant protein reduced a variety of quinones and napthoquinones, including several of allelopathic significance, using either NADH or NADPH as electron donors. The protein had an absorption spectrum consistent with it being a flavoprotein and was inhibited by the quinone reductase inhibitor dicumarol. We propose that the TvQR2 protein functions as a quinone reductase in plant roots to mitigate the toxicity of exogenous quinones in the rhizosphere.     

Redox and addition chemistry of quinoid compounds and its biological implications

The overall biological activity of quinones is a function of the physico-chemical properties of these compounds, which manifest themselves in a critical bimolecular reaction with bioconstituents. Attempts have been made to characterize this bimolecular reaction as a function of the redox properties of quinones in relation to hydrophobic or hydrophilic environments. The inborn physico-chemical properties of quinones are discussed on the basis of their reduction potential and dissociation constants, as well as the effect of environmental factors on these properties. Emphasis is given on the effect of methyl-, methoxy-, hydroxy-, and glutathionyl substituents on the reduction potential of quinones and the subsequent electron transfer processes. The redox chemistry of quinoid compounds is surveyed in terms of a) reactions involving only electron transfer, as those accomplished during the enzymic reduction of quinones and the non-enzymic interaction with redox couples generating semiquinones, and b) nucleophilic addition reactions. The addition of nucleophiles, entailing either oxidation or reduction of the quinone, are exemplified in reactions with oxygen- or sulfur nucleophiles, respectively. The former yields quinone epoxides, whereas the latter yields thioether-hydroquinone adducts as primary molecular products. The subsequent chemistry of these products is examined in terms of enzymic reduction, autoxidation, cross-oxidation, disproportionation, and free radical interactions. The detailed chemical mechanisms by which quinoid compounds exert cytotoxic, mutagenic and carcinogenic effects are considered individually in relation to redox cycling, alterations of thiol balance and Ca++ homeostasis, and covalent binding.     

o-quinone/quinone methide isomerase: a novel enzyme preventing the destruction of self-matter by phenoloxidase-generated quinones during immune response in insects

Melanization and encapsulation of invading foreign organisms observed during the immune response in insects is known to be due to the action of activated phenoloxidase. Phenoloxidase-generated quinones are deposited either directly or after self-polymerization on foreign objects accounting for the observed reactions. Since the reactions of quinones are nonenzymatic, they do not discriminate self from nonself and hence will also destroy self-matter. In this report we present evidence for the presence of a novel quinone/quinone methide isomerase in the hemolymph of Sarcophaga bullata which destroys long-lived quinones and hence acts to protect the self-matter. Quinone methides, formed by the action of this enzyme on physiologically important quinones, being unstable undergo rapid hydration to form nontoxic metabolites.     

Reversal of quinone-induced chlorophyll fluorescence quenching

We have used two methods to investigate the reversibility of the interaction of substituted quinones with the thylakoid membrane of plant chloroplasts. Treatment of chloroplasts with added quinones lowers the room-temperature Photosystem II chlorophyll fluorescence intensity by variable amounts depending on the identity and concentration of the quinone. The extent of restoration of the chlorophyll fluorescence level is used as a measure of the effectiveness of the reversal technique. One reversal method involves the addition of thiols to quinone-treated chloroplasts to alter the quinone in a chemical way via a nucleophilic 1,4-Michael addition. In general, the modified quinones exhibit a lower affinity for the thylakoid membrane, as evidenced by an accompanying increase in chlorophyll fluorescence. The thiol concentrations necessary for quenching reversal are found to be in the order [dithiothreitol] less than [2-mercaptoethanol] less than [glutathione]. The second reversal method examines the extent to which added quinones can be removed from thylakoid membranes using a concentration gradient established by resuspension of quinone-treated chloroplasts in quinone-free media. The results further support the reversible nature of the quinone inhibition and indicate that the extent of recovery is dependent upon the degree of fluorescence inhibition originally induced by the added quinone.     

Functional consequences of the organization of the photosynthetic apparatus in Rhodobacter sphaeroides. I. Quinone domains and excitation transfer in chromatophores and reaction center.antenna complexes

The purpose of this study was to gain information on the functional consequences of the supramolecular organization of the photosynthetic apparatus in the bacterium Rhodobacter sphaeroides. Isolated complexes of the reaction center (RC) with its core antenna ring (light-harvesting complex 1 (LH1)) were studied in their dimeric (native) form or as monomers with respect to excitation transfer and distribution of the quinone pool. Similar issues were examined in chromatophore membranes. The relationship between the fluorescence yield and the amount of closed centers is indicative of a very efficient excitation transfer between the two monomers in isolated dimeric complexes. A similar dependence was observed in chromatophores, suggesting that excitation transfer in vivo from a closed RC.LH1 unit is also essentially directed to its partner in the dimer. The isolated complexes were found to retain 25-30% of the endogenous quinone acceptor pool, and the distribution of this pool among the complexes suggests a cooperative character for the association of quinones with the protein complexes. In chromatophores, the decrease in the amount of photoreducible quinones when inhibiting a fraction of the centers implies a confinement of the quinone pool over small domains, including one to six reaction centers. We suggest that the crowding of membrane proteins may not be the sole reason for quinone confinement and that a quinone-rich region is formed around the RC.LH1 complexes.     

Thiols oxidation and covalent binding of BSA by cyclolignanic quinones are enhanced by the magnesium cation

A novel cyclolignanic quinone, 7-acetyl-3',4'-didemethoxy-3',4'-dioxopodophyllotoxin (CLQ), inhibits topoisomerase II (TOPO II) activity. The extent of this inhibition was greater than that produced by the etoposide quinone (EQ) or etoposide. Glutathione (GSH) reduces EQ and CLQ to their corresponding semiquinones under anaerobic conditions. The latter were detected by EPR spectroscopy in the presence of MgCl₂ but not in its absence. Semiquinone EPR spectra change with quinone/GSH mol ratio, suggesting covalent binding of GSH to the quinones. Quinone-GSH covalent adducts were isolated and identified by ESI-MS. These orthoquinones also react with nucleophilic groups from BSA to bind covalently under anaerobic conditions. BSA thiol consumption and covalent binding by these quinones are enhanced by MgCl₂. Complex formation between the parent quinones and Mg⁺² was also observed. Density functional calculations predict the observed blue-shifts in the absorption spectra peaks and large decreases in the partial negative charge of electrophilic carbons at the quinone ring when the quinones are complexed to Mg⁺². These observations suggest a possible role of Mg⁺² chelation by these quinones in increasing TOPO II thiol and/or amino/imino reactivity with these orthoquinones.     

Dynamics and diffusion in photosynthetic membranes from rhodospirillum photometricum

Photosynthetic organisms drive their metabolism by converting light energy into an electrochemical gradient with high efficiency. This conversion depends on the diffusion of quinones within the membrane. In purple photosynthetic bacteria, quinones reduced by the reaction center (RC) diffuse to the cytochrome bc(1) complex and then return once reoxidized to the RC. In Rhodospirillum photometricum the RC-containing core complexes are found in a disordered molecular environment, with fixed light-harvesting complex/core complex ratio but without a fixed architecture, whereas additional light-harvesting complexes synthesized under low-light conditions pack into large paracrystalline antenna domains. Here, we have analyzed, using time-lapse atomic force microscopy, the dynamics of the protein complexes in the different membrane domains and find that the disordered regions are dynamic whereas ordered antennae domains are static. Based on our observations we propose, and analyze using Monte Carlo simulations, a model for quinone diffusion in photosynthetic membranes. We show that the formation of large static antennae domains may represent a strategy for increasing electron transfer rates between distant complexes within the membrane and thus be important for photosynthetic efficiency.     

Effect Of Glutathione On The Redox Transitions Of Naphthohydroquinone Derivatives Formed During Dt-Diaphorase Catalysis

The oxidation of GSH coupled to the redox transitions of 1, Cnaphthoquinone derivatives during DT-diaphorase catalysis was examined. The quinones studied included 1,4-naphthoquinone and its dimethoxy-and hydroxy derivatives and were selected according to their different ability to undergo nucleophilic addition with GSH and the dual effect of superoxide dismutase on hydroquinone autoxidation

GSH was oxidized to GSSG during the redox transitions of the above quinones, regardless of their substitution pattern. This effect was accompanied by an increase of total O₂ consumption, indicating the ability of GSH to support quinone redox cycling. The values for the relationship [O₂]_consumed[GSSG]_formde were, with every quinone examined, above unity. thus pointing to the occurrence of autoxidation reactions other than those involved during GSSG formation

These results are discussed in terms of the functional group chemistry of the quinones and the ther-modynamic properties of the reactions involved in the reduction of the semi- to the hydro-quinone by GSH     

Thiols oxidation and covalent binding of BSA by cyclolignanic quinones are enhanced by the magnesium cation

A novel cyclolignanic quinone, 7-acetyl-3′,4′-didemethoxy-3′,4′-dioxopodophyllotoxin (CLQ), inhibits topoisomerase II (TOPO II) activity. The extent of this inhibition was greater than that produced by the etoposide quinone (EQ) or etoposide. Glutathione (GSH) reduces EQ and CLQ to their corresponding semiquinones under anaerobic conditions. The latter were detected by EPR spectroscopy in the presence of MgCl₂ but not in its absence. Semiquinone EPR spectra change with quinone/GSH mol ratio, suggesting covalent binding of GSH to the quinones. Quinone-GSH covalent adducts were isolated and identified by ESI-MS. These orthoquinones also react with nucleophilic groups from BSA to bind covalently under anaerobic conditions. BSA thiol consumption and covalent binding by these quinones are enhanced by MgCl₂. Complex formation between the parent quinones and Mg⁺² was also observed. Density functional calculations predict the observed blue-shifts in the absorption spectra peaks and large decreases in the partial negative charge of electrophilic carbons at the quinone ring when the quinones are complexed to Mg⁺². These observations suggest a possible role of Mg⁺² chelation by these quinones in increasing TOPO II thiol and/or amino/imino reactivity with these orthoquinones.     

An x-ray absorption study of the iron site in bacterial photosynthetic reaction centers.

Measurements were made of the extended x-ray absorption fine structure (EXAFS) of the iron site in photosynthetic reaction centers from the bacterium Rhodopseudomonas sphaeroides. Forms with two quinones, two quinones with added o-phenanthroline, and one quinone were studied. Only the two forms containing two quinones maintained their integrity and were analyzed. The spectra show directly that the added o-phenanthroline does not chelate the iron atom. Further analysis indicates that the iron is octahedrally coordinated by nitrogen and/or oxygen atoms located at various distances, with the average value of about 2.14 A. The analysis suggests that most of the ligands are nitrogens and that three of the nitrogen ligands belong to histidine rings. This interpretation accounts for several unusual features of the EXAFS spectrum. We speculate that the quinones are bound to the histidine rings in some manner. Qualitative features of the absorption edge spectra also are discussed and are related to the Fe-ligand distance.     

Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study

Since the higher redox potential of quinone molecules has been correlated with enhanced cellular deleterious effects, we studied the ability of the association of ascorbate with several quinones derivatives (having different redox potentials) to cause cell death in K562 human leukaemia cell line. The rationale is that the reduction of quinone by ascorbate should be dependent of the quinone half-redox potential thus determining if reactive oxygen species (ROS) are formed or not, leading ultimately to cell death or cell survival. Among different ROS that may be formed during redox cycling between ascorbate and the quinone, the use of different antioxidant compounds (mannitol, desferal, N-acetylcysteine, catalase and superoxide dismutase) led to support H2O2 as the main oxidizing agent. We observed that standard redox potentials, oxygen uptake, free ascorbyl radical formation and cell survival were linked. The oxidative stress induced by the mixture of ascorbate and the different quinones decreases cellular contents of ATP and GSH while caspase-3-like activity remains unchanged. Again, we observed that quinones having higher values of half-redox potential provoke a severe depletion of ATP and GSH when they were associated with ascorbate. Such a drop in ATP content may explain the lack of activation of caspase-3. In conclusion, our results indicate that the cytotoxicity of the association quinone/ascorbate on K562 cancer cells may be predicted on the basis of half-redox potentials of quinones.