Reactivity of ubiquinone and ubiquinol with superoxide and the hydroperoxyl radical: implications for in vivo antioxidant activity

Endogenous ubiquinones (UQ) such as coenzyme Q(10) are essential electron carriers in the mitochondrial respiratory chain, and the reduced ubiquinol form (UQH(2)) is a chain-breaking antioxidant, decreasing oxidative damage caused by lipid peroxidation within mitochondria. Consequently, exogenous UQ are used as therapies to decrease mitochondrial oxidative damage. The proximal radical produced during mitochondrial oxidative stress is superoxide (O(2)(.-)) and the reaction between UQ and O(2)(.-) to form the ubisemiquinone radical anion (UQ(.-)) may also be important for the scavenging of O(2)(.-) by exogenous UQ. The situation in vivo is that many UQ are predominantly located in the hydrophobic membrane core, from which O(2)(.-) will be excluded but its conjugate acid, HOO(.), can enter. The reactivity of UQ or UQH(2) with HOO(.) has not been reported previously. Here a pulse radiolysis study on the reactions between UQ/UQH(2) and O(2)(.-)/HOO(.) in water and in solvent systems mimicking the surface and core of biological membranes has been undertaken. O(2)(.-) reacts very rapidly with UQ, suggesting that this may contribute to the scavenging of O(2)(.-) in vivo. In contrast, UQH(2) reacts relatively slowly with HOO(.), but rapidly with other oxygen- and carbon-centered radicals, indicating that the antioxidant role of UQH(2) is mainly in preventing lipid peroxidation.     

A novel ubiquinone reductase activity in rat cytosol.

Ubiquinone (UQ) reductase activity which reduces UQ to ubiquinol (UQH2) in rat tissues was roughly proportional to the UQH2/total UQ ratio in respective tissues. The highest activity was found in the liver, showing the highest UQH2/total UQ ratio. A greater part of liver UQ reductase activity was located in the cytosol. Within a week, the liver UQ reductase activity decreased by 80% even at -20 degrees C. The DT-diaphorase activity was stable. UQ reductase required NADPH as the hydrogen donor and was not inhibited by a less than 1 microM concentration of dicoumarol. There was no stimulation of UQ reductase in the presence of bovine serum albumin nor in Triton X-100. Yet, both stimulated DT-diaphorase. As a result, UQ reductase appeared to be a novel NADPH-UQ oxidoreductase and responsible for the UQ redox state in liver.     

Cytosolic NADPH-UQ reductase, the enzyme responsible for cellular ubiquinone redox cycle as an endogenous antioxidant in the rat liver.

Cellular ubiquinone (UQ) is expected to act as an endogenous antioxidant against oxidative stress. To confirm this, UQ-reductases which are necessary to regenerate ubiquinol (UQH2) were investigated in rat tissue, and a novel NADPH-dependent UQ (NADPH-UQ) reductase was found in cytosol. The cytosolic NADPH-UQ reductase activity accounted for more than 80% of UQ-10 reduction by the rat liver homogenate in the presence of NADPH. Furthermore, the NADPH-UQ reductase activities in various tissues were correlated to the redox states of UQ in the corresponding tissues. Rat liver cytosol with NADPH protected lecithin liposomes containing UQ-10, as well as UQH2-10 from AMVN (2,2'-azobis(2,4-dimethylvaleronitrile))-induced lipid peroxidation. The enzyme purified from rat liver cytosol, reduced UQ-10 in lecithin liposomes at approximately the same rate as did cytosol. These results supported that cytosolic NADPH-UQ reductase is the enzyme responsible for nonmitochondrial UQ reduction acting as an endogenous antioxidant against oxidative stress. The antioxidant role of the UQ redox cycle and NADPH-UQ reductase was discussed in relation to other cellular NADPH-dependent antioxidant enzymes.     

Electron acceptors of bacterial photosynthetic reaction centers. II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex.

The photoreduction of ubiquinone in the electron acceptor complex (QIQII) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H+ in the reduction was revealed by the pH dependence of the electron transfer events and by net H+ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash QII receives an electron via QI to form a stable ubisemiquinone anion (QII-); the second flash generates QI-. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H+, to produce QIIH2. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H+ uptake. Above pH 6 there is a progressive increase in H+ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H+ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to QII-. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H+ binding electron transfer following the second flash.     

Amino acid residues interacting with both the bound quinone and coenzyme, pyrroloquinoline quinone, in Escherichia coli membrane-bound glucose dehydrogenase

The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e(aq)(-)) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e(aq)(-), although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both with the bound UQ and PQQ in mGDH.     

Are Reduced Ubiquinones Oxygen Radical Generators?

Ubiquinone (UQ) is the only natural compound which was reported both to generate and to scavenge oxygen-derived radicals. Redox-cycling of ubiquinone may yield six different species of the parent compound: UQH₂, UQH⁻, UQ²⁻, UQH^•, UQ^•−, and UQ. Ubiquinol (UQH₂) is unequivocally considered to be the ubiquinone species capable of scavenging oxygen-derived radicals. In contrast, the ubiquinone species responsible for one e⁻reduction of dioxygen (O₂) thereby initiating the cascade of oxidative stress is still a matter of controversial debate. In the present study this question was approached by following the effect of O₂on the stability of the various reduced forms of UQ. For this purpose conditions were designed allowing the selective accumulation of the two protonated and of the two deprotonated forms of reduced ubiquinones. Our results exclude both protonated (ubiquinol, UQH₂) and anionic (ubiquinol anion, UQH⁻, and ubiquinol dianion, UQ²⁻) fully reduced ubiquinones as the source exerting one e⁻reduction of O₂. Ubisemiquinone (semiquinone radical, UQH^•), when protonated, underwent rapid disproportionation, while transition to the semireduced anionic form (semiquinone radical anion, UQ^•−) was found to favor autoxidation. The results obtained in this study provide a chemical base for the assessment of one e⁻transfer from redox-cycling UQ to O₂in the respiratory chain and in biomembranes where ubihydroquinol is suggested to exert antioxidant activities.     

Crystal structures of acid blue and alkaline purple forms of bacteriorhodopsin

Bacteriorhodopsin, a light-driven proton pump found in the purple membrane of Halobacterium salinarum, exhibits purple at neutral pH but its color is sensitive to pH. Here, structures are reported for an acid blue form and an alkaline purple form of wild-type bacteriorhodopsin. When the P622 crystal prepared at pH 5.2 was acidified with sulfuric acid, its color turned to blue with a pKa of 3.5 and a Hill coefficient of 2. Diffraction data at pH 2-5 indicated that the purple-to-blue transition accompanies a large structural change in the proton release channel; i.e. the extracellular half of helix C moves towards helix G, narrowing the proton release channel and expelling a water molecule from a micro-cavity in the vicinity of the retinal Schiff base. In this respect, the acid-induced structural change resembles the structural change observed upon formation of the M intermediate. But, the acid blue form contains a sulfate ion in a site(s) near Arg82 that is created by re-orientations of the carboxyl groups of Glu194 and Glu204, residues comprising the proton release complex. This result suggests that proton uptake by the proton release complex evokes the anion binding, which in turn induces protonation of Asp85, a key residue regulating the absorption spectrum of the chromophore. Interestingly, a pronounced structural change in the proton release complex was also observed at high pH; i.e. re-orientation of Glu194 towards Tyr83 was found to take place at around pH 10. This alkaline transition is suggested to be accompanied by proton release from the proton release complex and responsible for rapid formation of the M intermediate at high pH.     

EPR spin trapping detection of carbon-centered carotenoid and beta-ionone radicals

Free radical intermediates were detected by the electron paramagnetic resonance spin trapping technique upon protonation/deprotonation reactions of carotenoid and beta-ionone radical ions. The hyperfine coupling constants of their spin adducts obtained by spectral simulation indicate that carbon-centered radicals were trapped. The formation of these species was shown to be a result of chemical oxidation of neutral compounds by Fe(3+) or I(2) followed by deprotonation of the corresponding radical cations or addition of nucleophilic agents to them. Bulk electrolysis reduction of beta-ionone and carotenoids also leads to the formation of free radicals via protonation of the radical anions. Two different spin adducts were detected in the reaction of carotenoid polyenes with piperidine in the presence of 2-methyl-2-nitroso-propane (MNP). One is attributable to piperidine radicals (C(5)H(10)N*) trapped by MNP and the other was identified as trapped neutral carotenoid (beta-ionone) radical produced via protonation of the radical anion. Formation of these radical anions was confirmed by ultraviolet-visible spectroscopy. It was found that the ability of carotenoid radical anions/cations to produce neutral radicals via protonation/deprotonation is more pronounced for unsymmetrical carotenoids with terminal electron-withdrawing groups. This effect was confirmed by the radical cation deprotonation energy (H(D)) estimated by semiempirical calculations. The results indicate that the ability of carotenoid radical cations to deprotonate decreases in the sequence: beta-ionone > unsymmetrical carotenoids > symmetrical carotenoids. The minimum H(D) values were obtained for proton abstraction from the C(4) atom and the C(5)-methyl group of the cyclohexene ring. It was assumed that deprotonation reaction occurs preferentially at these positions.     

Gel-phase microdomains and lipid rafts in monolayers affect the redox properties of ubiquinone-10

The redox properties of ubiquinone-10 (UQ) were examined in monolayers of mixtures of dioleoylphosphatidylcholine, palmitoylsphingomyelin, and cholesterol of different compositions, self-assembled on a mercury electrode, over the pH range from 7.5 to 9.5. A detailed analysis of the cyclic voltammograms of UQ in the above lipid environments points to a mechanism consisting of an elementary electron transfer step followed by two protonation (or deprotonation) steps in quasiequilibrium and by a further electron transfer step. In a lipid environment of solid-ordered (s_o) microdomains in a liquid-disordered (l_d) matrix, electron transport across the lipid monolayer takes place in the l_d phase. In a pure s_o phase, UQ tends to segregate into UQ-rich pools, exhibiting reversible electron transfer steps. In a lipid environment consisting of liquid-ordered (l_o) microdomains (lipid rafts) in an l_d matrix, UQ molecules tend to localize along the edge of the lipid rafts. However, in a lipid environment consisting exclusively of l_o and s_o microdomains, UQ molecules tend to segregate into UQ-rich pools. In all lipid environments, electron transport by UQ occurs with the quinone moiety localized on the solution side with respect to the ester linkages of the dioleoylphosphatidylcholine molecules.     

Characterization of cellular uptake and distribution of coenzyme Q10 and vitamin E in PC12 cells

Coenzyme Q (CoQ) is a well-known electron transporter in the mitochondrial respiratory chain. Furthermore, ubiquinol (UQH(2))--a reduced form of ubiquinone (UQ)--has been shown to act as a radical-scavenging antioxidant. Some studies have reported the beneficial effect of CoQ addition to cultured cells; however, the cellular uptake and distribution of CoQ have not been elucidated. In the present study, we used rat pheochromocytoma PC12 cells to investigate and compare the cellular uptake and distribution of CoQ(10) and alpha-tocopherol (alphaT). UQ(10) or UQ(10)H(2) treatment resulted in an increase in the cellular content of both CoQ(10) in a time- and concentration-dependent manner. A subcellular fractionation study revealed that the added UQ(10) as well as UQ(10)H(2) mainly localized in the mitochondrial fraction, which is similar to the localization of endogenous CoQ but different from that of alphaT. The cellular distribution of alphaT directly corresponded to the lipid distribution, while the CoQ distribution did not show any relationship with the lipid distribution, particularly in the mitochondrial and microsomal fractions. These results indicate that the cellular distribution of CoQ is completely different from that of alphaT; moreover, a certain system which accumulates CoQ preferentially in mitochondria may be suggested.     

The multiple functions of coenzyme Q

The coenzyme function of ubiquinone was subject of extensive studies in mitochondria since more than 40 years. The catalytic activity of ubiquinone (UQ) in electron transfer and proton translocation in cooperation with mitochondrial dehydrogenases and cytochromes contributes essentially to the bioenergetic activity of ATP synthesis. In the past two decades UQ was recognized to exert activities which differ from coenzyme functions in mitochondria. From extraction/reincorporation experiments B. Chance has drawn the conclusion that redox-cycling of mitochondrial ubiquinone supplies electrons for univalent reduction of dioxygen. The likelihood of O2(.-) release as normal byproduct of respiration was based on the existence of mitochondrial SOD and the fact that mitochondrial oxygen turnover accounts for more than 90% of total cellular oxygen consumption. Arguments disproving this concept are based on results obtained from a novel noninvasive, more sensitive detection method of activated oxygen species and novel experimental approaches, which threw light into the underlying mechanism of UQ-mediated oxygen activation. Single electrons for O2(.-) formation are exclusively provided by deprotonated ubisemiquinones. Impediment of redox-interaction with the bc1 complex in mitochondria or the lack of stabilizing interactions with redox-partners are promotors of autoxidation. The latter accounts for autoxidation of antioxidant-derived ubisemiquinones in biomembranes, which do not recycle oxidized ubiquinols. Also O2(.-)-derived H2O2 was found to interact with ubisemiquinones both in mitochondria and nonrecycling biomembranes when ubiquinol was active as antioxidant. The catalysis of reductive homolytic cleavage of H2O2, which contributes to HO. formation in biological systems was confirmed under defined chemical conditions in a homogenous reduction system. Apart from dioxygen and hydrogen peroxide we will provide evidence that also nitrite may chemically interact with the ubiquinol/bc1 redox couple in mitochondria. The reaction product NO was reported elsewhere to be a significant bioregulator of the mitochondrial respiration and O2 activation. Another novel finding documents the bioenergetic role of UQ in lysosomal proton intransport. A lysosomal chain of redox couples will be presented, which includes UQ and which requires oxygen as the terminal electron acceptor.     

Critical aspects of the antioxidant function of coenzyme Q in biomembranes.

Coenzyme Q (ubiquinone, UQ) is increasingly considered as a significant natural antioxidant, which protects biomembranes in concert with alpha-tocopherol. In vitro experiments demonstrated that reduced UQ (ubiquinol) can improve the chain-breaking activities of alpha-tocopherol by recycling the antioxidant-derived reaction product, the chromanoxyl radical, to the native antioxidant. Less attention, however, was devoted to the antioxidant-derived reaction products of reduced UQ. Although both alpha-tocopherol and ubiquinol were found to be equally effective in scavenging chain-propagating lipid radicals. alpha-tocopherol protected lipid membranes from lipid peroxidation more efficiently than ubiquinol. The present study not only provides data which document this discrepancy but also contributes experimental data on the existence of ubiquinol derived pro-oxidants, which give an explanation of this phenomenon.     

Superoxide reductase from Desulfoarculus baarsii: identification of protonation steps in the enzymatic mechanism

Superoxide reductase (SOR) is a metalloenzyme that catalyzes the reduction of O2*- to H2O2 and provides an antioxidant mechanism in some anaerobic and microaerophilic bacteria. Its active site contains an unusual mononuclear ferrous center (center II). Protonation processes are essential for the reaction catalyzed by SOR, since two protons are required for the formation of H2O2. We have investigated the acido-basic and pH dependence of the redox properties of the active site of SOR from Desulfoarculus baarsii, both in the absence and in the presence of O2*-. In the absence of O2*-, the reduction potential and the absorption spectrum of the iron center II exhibit a pH transition. This is consistent with the presence of a base (BH) in close proximity to the iron center which modulates its reduction properties. Studies of mutants of the closest charged residues to the iron center II (E47A and K48I) show that neither of these residues are the base responsible for the pH transitions. However, they both interact with this base and modulate its pKa value. By pulse radiolysis, we confirm that the reaction of SOR with O2*- involves two reaction intermediates that were characterized by their absorption spectra. The precise step of the catalytic cycle in which one protonation takes place was identified. The formation of the first reaction intermediate, from a bimolecular reaction of SOR with O2*-, does not involve proton transfer as a rate-limiting step, since the rate constant k1 does not vary between pH 5 and pH 9.5. On the other hand, the rate constant k2 for the formation of the second reaction intermediate is proportional to the H+ concentration in solution, suggesting that the proton arises directly from the solvent. In fact, BH, E47, and K48 have no role in this step. This is consistent with the first intermediate being an iron(III)-peroxo species and the second one being an iron(III)-hydroperoxo species. We propose that BH may be involved in the second protonation process corresponding to the release of H2O2 from the iron(III)-hydroperoxo species.     

Demonstration of a collisional interaction of ubiquinol with the ubiquinol-cytochrome c2 oxidoreductase complex in chromatophores from Rhodobacter sphaeroides

Ubiquinone-10 can be extracted from lyophilized chromatophores of Rhodobacter sphaeroides (previously called Rhodopseudomonas sphaeroides) without significant losses in other components of the electron-transfer chain or irreversible damages in the membrane structure. The pool of ubiquinone can be restored with exogenous UQ-10 to sizes larger than the ones in unextracted membranes. The decrease in the pool size has marked effects on the kinetics of reduction of cytochrome b-561 induced by a single flash of light and measured in the presence of antimycin. The initial rate of reduction, which in unextracted preparations increases on reduction of the suspension over the Eh range between 170 and 100 mV (pH 7), is also stimulated in partially UQ-depleted membranes, although at more negative Eh's. When the UQ pool is completely extracted the rate of cytochrome (Cyt) b-561 reduction is low and unaffected by the redox potential. In membranes enriched in UQ-10 above the physiological level the titration curve of the rate of Cyt b-561 reduction is displaced to Eh values more positive than in controls. This effect is saturated when the size of the UQ pool is about 2-3 times larger than the native one. The reduction of Cyt b-561 always occurs a short time after the flash is fired; also the duration of this lag is dependent on Eh and on the size of the UQ pool. A decrease or an increase in the pool size causes a displacement of the titration curve of the lag to more negative or to more positive Eh's, respectively. Similarly, the lag becomes Eh independent and markedly longer than in controls when the pool is completely extracted. These results demonstrate that the rate of turnover of the ubiquinol oxidizing site in the b-c1 complex depends on the actual concentration of ubiquinol present in the membrane and that ubiquinol from the pool is oxidized at this site with a collisional mechanism. Kinetic analysis of the data indicates that this reaction obeys a Michaelis-Menten type equation, with a Km of 3-5 ubiquinol molecules per reaction center.     

Proton transfer reactions associated with the reaction of the fully reduced,purified cytochrome C oxidase with molecular oxygen and ferricyanide

A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from approximately 3.0 H(+)/COX at pH 6.2-6.3 to approximately 1.2 H(+)/COX at pH 8.0-8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from approximately 1.0 H(+)/COX at pH 6.2-6.3 to approximately 2.8 H(+)/COX at pH 8.0-8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3-1.6 H(+)/COX in the pH range 6.2-8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O(2) to 2H(2)O. The results show that the expected stoichiometric proton consumption of 4H(+) in the reduction of O(2) to 2H(2)O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H(+)/COX are initially taken up in the reduction of O(2) to two OH(-) groups bound to the binuclear Fe a(3)-Cu(B) center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H(2)O. At alkaline pHs the third and fourth protons are taken up with formation of 2H(2)O only upon rereduction of COX.     

Structure of the yeast cytochrome bc1 complex with a hydroxyquinone anion Qo site inhibitor bound

Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc1 complex catalysis. Binding of the competitive inhibitor 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole to the Qo site of the cytochrome bc1 complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pKa = 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His181 and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr279. The hydroxy group of the latter contributes to stabilization of the Rieske protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His181 with a pKa of 7.5. Glu272, a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme bL propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Qo site.     

Electron acceptors of bacterial photosynthetic reaction centers II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex

The photoreduction of ubiquinone in the electron acceptor complex (Q₁Q₁₁) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H⁺ in the reduction was revealed by the pH dependence of the electron transfer events and by net H⁺ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash Q₁₁ receives an electron via Q₁ to form a stable ubisemiquinone anion (Q?^?₁₁); the second flash generates Q?^?₁. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H⁺, to produce Q₁₁H₂. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H⁺ uptake. Above pH 6 there is a progressive increase in H⁺ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H⁺ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to Q?^?₁₁. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H⁺ binding electron transfer following the second flash.     

Comparative study on dynamics of antioxidative action of alpha-tocopheryl hydroquinone, ubiquinol, and alpha-tocopherol against lipid peroxidation.

Alpha-tocopheryl quinone is a metabolite of alpha-tocopherol (TOH) in vivo. The antioxidant action of its reduced form, alpha-tocopheryl hydroquinone (TQH2), has received much attention recently. In the present study, the antioxidative activity of TQH2 was studied in various systems in vitro and compared with that of ubiquinol-10 (UQH2) or TOH to obtain the basic information on the dynamics of the antioxidant action of TQH2. First, their hydrogen-donating abilities were investigated in the reaction with galvinoxyl, a stable phenoxyl radical, and TQH2 was found to possess greater second-order rate constant (1.0 x 10(4) M(-1) s(-1)) than UQH2 (6.0 x 10(3) M(-1) s(-1)) and TOH (2.4 x 10(3) M(-1) s(-1)) at 25 degrees C in ethanol. The stoichiometric numbers were obtained as 1.9, 2.0, and 1.0 for TQH2, UQH2, and TOH, respectively, in reducing galvinoxyl. Second, their relative reactivities toward peroxyl radicals were assessed in competition with N,N'-diphenyl-p-phenylenediamine (DPPD) and found to be 6.0 (TQH2), 1.9 (UQH2), and 1.0 (TOH). Third, their antioxidant efficacies were evaluated in the oxidation of methyl linoleate in organic solvents and in aqueous dispersions. The antioxidant potency decreased in the order TOH > UQH2 > TQH2, as assessed by either the extent of the reduction in the rate of oxidation or the duration of inhibition period. The reverse order of their reactivities toward radicals and their antioxidant efficacies was interpreted by the rapid autoxidation of TQH2 and UQH2, carried out by hydroperoxyl radicals. Although neither TQH2 nor UQH2 acted as a potent antioxidant by itself, they acted as potent antioxidants in combination with TOH. TQH2 and UQH2 reduced alpha-tocopheroxyl radical to spare TOH, whereas TOH suppressed the autoxidation of TQH2 and UQH2. In the micelle oxidation, the antioxidant activities of TQH2, UQH2, and TOH were similar, whereas 2,2,5,7,8-pentamethyl-6-chromanol exerted much more potent efficacy than TQH2, UQH2, or TOH. These results clearly show that the antioxidant potencies against lipid peroxidation are determined not only by their chemical reactivities toward radicals, but also by the fate of an antioxidant-derived radical and the mobility of the antioxidant at the microenvironment.     

Transient formation of a neutral ubisemiquinone radical and subsequent intramolecular electron transfer to pyrroloquinoline quinone in the Escherichia coli membrane-integrated glucose dehydrogenase

The membrane-bound quinoprotein glucose dehydrogenase (mGDH) in Escherichia coli contains pyrroloquinoline quinone (PQQ) and participates in the direct oxidation of D-glucose to D-gluconate by transferring electrons to ubiquinone (UQ). To elucidate the mechanism of ubiquinone reduction by mGDH, we applied a pulse radiolysis technique to mGDH with or without bound UQ8. With the UQ8-bound enzyme, a hydrated electron reacted with mGDH to form a transient species with an absorption maximum at 420 nm, characteristic of formation of a neutral ubisemiquinone radical. Subsequently, the decay of the absorbance at 420 nm was accompanied by an increase in the absorbance at 370 nm. Experiments with the PQQ-free apoenzyme showed no such subsequent absorption changes, although ubisemiquinone was formed. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone radical at the UQ8 binding site to PQQ exists in mGDH. The first-order rate constant of this process was calculated to be equal to 1.2 x 10(3) s(-1). These findings are consistent with our proposal that during the catalytic cycle of mGDH the bound UQ8 mediates electron transfer from the reduced PQQ to UQ8 pools.     

Menaquinone as well as ubiquinone as a bound quinone crucial for catalytic activity and intramolecular electron transfer in Escherichia coli membrane-bound glucose dehydrogenase

Escherichia coli membrane-bound glucose dehydrogenase (mGDH), which is one of quinoproteins containing pyrroloquinoline quinone (PQQ) as a coenzyme, is a good model for elucidating the function of bound quinone inside primary dehydrogenases in respiratory chains. Enzymatic analysis of purified mGDH from cells defective in synthesis of ubiquinone (UQ) and/or menaquinone (MQ) revealed that Q-free mGDH has very low levels of activity of glucose dehydrogenase and UQ2 reductase compared with those of UQ-bearing mGDH, and both activities were significantly increased by reconstitution with UQ1. On the other hand, MQ-bearing mGDH retains both catalytic abilities at the same levels as those of UQ-bearing mGDH. A radiolytically generated hydrated electron reacted with the bound MQ to form a semiquinone anion radical with an absorption maximum at 400 nm. Subsequently, decay of the absorbance at 400 nm was accompanied by an increase in the absorbance at 380 nm with a first order rate constant of 5.7 x 10(3) s(-1). This indicated that an intramolecular electron transfer from the bound MQ to the PQQ occurred. EPR analysis revealed that characteristics of the semiquinone radical of bound MQ are similar to those of the semiquinone radical of bound UQ and indicated an electron flow from PQQ to MQ as in the case of UQ. Taken together, the results suggest that MQ is incorporated into the same pocket as that for UQ to perform a function almost equivalent to that of UQ and that bound quinone is involved at least partially in the catalytic reaction and primarily in the intramolecular electron transfer of mGDH.