Transient formation of ubisemiquinone radical and subsequent electron transfer process in the Escherichia coli cytochrome bo

To elucidate a unique mechanism for the quinol oxidation in the Escherichia coli cytochrome bo, we applied pulse radiolysis technique to the wild-type enzyme with or without a single bound ubiquinone-8 at the high-affinity quinone binding site (Q(H)), using N-methylnicotinamide (NMA) as an electron mediator. With the ubiquinone bound enzyme, the reduction of the oxidase occurred in two phases as judged from kinetic difference spectra. In the faster phase, the transient species with an absorption maximum at 440 nm, a characteristic of the formation of ubisemiquinone anion radical, appeared within 10 micros after pulse radiolysis. In the slower phase, a decrease of absorption at 440 nm was accompanied by an increase of absorption at 428 and 561 nm, characteristic of the reduced form. In contrast, with the bound ubiquinone-8-free wild-type enzyme, NMA radicals directly reduced hemes b and o, though the reduction yield was low. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone anion radical at the Q(H) site to heme b exists in cytochrome bo. The first-order rate constant of this process was calculated to be 1.5 x 10(3) s(-1) and is comparable to a turnover rate for ubiquinol-1. The rate constant for the intramolecular electron transfer decreased considerably with increasing pH, though the yields of the formation of ubisemiquinone anion radical and the subsequent reduction of the hemes were not affected. The pH profile was tightly linked to the stability of the bound ubisemiquinone in cytochrome bo [Ingledew, W. J., Ohnishi, T., and Salerno, J. C. (1995) Eur. J. Biochem. 227, 903-908], indicating that electron transfer from the bound ubisemiquinone at the Q(H) site to the hemes slows down at the alkaline pH where the bound ubisemiquinone can be stabilized. These findings are consistent with our previous proposal that the bound ubiquinone at the Q(H) site mediates electron transfer from the low-affinity quinol oxidation site in subunit II to low-spin heme b in subunit I.     

Direct and mediated electron transfer between intact succinate:quinone oxidoreductase from Bacillus subtilis and a surface modified gold electrode reveals redox state-dependent conformational changes

Succinate:quinone oxidoreductase (SQR) from Bacillus subtilis consists of two hydrophilic protein subunits comprising succinate dehydrogenase, and a di-heme membrane anchor protein harboring two putative quinone binding sites, Q_p and Q_d. In this work we have used spectroelectrochemistry to study the electronic communication between purified SQR and a surface modified gold capillary electrode. In the presence of two soluble quinone mediators the midpoint potentials of both hemes were revealed essentially as previously determined by conventional redox titration (heme b_H, E_m = + 65 mV, heme b_L, E_m = − 95 mV). In the absence of mediators the enzyme still communicated with the electrode, albeit with a reproducible hysteresis, resulting in the reduction of both hemes occurring approximately at the midpoint potential of heme b_L, and with a pronounced delay of reoxidation. When the specific inhibitor 2-n-heptyl-4 hydroxyquinoline N-oxide (HQNO), which binds to Q_d in B. subtilis SQR, was added together with the two quinone mediators, rapid reductive titration was still possible which can be envisioned as an electron transfer occurring via the HQNO insensitive Q_p site. In contrast, the subsequent oxidative titration was severely hampered in the presence of HQNO, in fact it completely resembled the unmediated reaction. If mediators communicate with Q_p or Q_d, either event is followed by very rapid electron redistribution within the enzyme. Taken together, this strongly suggests that the accessibility of Q_p depended on the redox state of the hemes. When both hemes were reduced, and Q_d was blocked by HQNO, quinone-mediated communication via the Q_p site was no longer possible, revealing a redox-dependent conformational change in the membrane anchor domain.     

Effect of inhibitors,redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of ¹⁴C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q_B, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q_A to Q_B) occurs via displacement of UQ from the Q_B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q_B to Q^?_B but is practically unaffected by reduction of Q_A to Q^?_A in the absence of Q_B. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q_B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q_B 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.     

ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo3 quinol oxidase.

The putative oxidation of ubiquinol by the cytochrome bo3 terminal oxidase of Escherichia coli in sequential one-electron steps requires stabilization of the semiquinone. ENDOR spectroscopy has recently been used to study the native ubisemiquinone radical formed in the cytochrome bo3 quinone-binding site [Veselov, A.V., Osborne, J.P., Gennis, R.B. & Scholes, C.P. (2000) Biochemistry 39, 3169-3175]. Comparison of these spectra with those from the decyl-ubisemiquinone radical in vitro indicated that the protein induced large changes in the electronic structure of the ubisemiquinone radical. We have used quinone-substitution experiments to obtain ENDOR spectra of ubisemiquinone, phyllosemiquinone and plastosemiquinone anion radicals bound at the cytochrome bo3 quinone-binding site. Large changes in the electronic structures of these semiquinone anion radicals are induced on binding to the cytochrome bo3 oxidase. The changes in electronic structure are, however, independent of the electronic structures of these semiquinones in vitro. Thus it is shown to be the structure of this binding site in the protein, not the covalent structure of the bound quinone, that determines the electronic structure of the protein-bound semiquinone.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

A primary respiratory Na+ pump of an anaerobic bacterium: the Na+-dependent NADH:quinone oxidoreductase of Klebsiella pneumoniae

Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na⁺ or Li⁺ ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaCl addition. Inverted bacterial membrane vesicles accumulated Na⁺ ions upon NADH oxidation. Na⁺ uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na⁺ pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by O₂, but by ferricyanide or menadione in a Na⁺-independent manner. The Na⁺-dependent NADH oxidation by O₂ was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaCl and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaCl, but not with NaCl and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na⁺-independent reaction sequence and then dismutate in a Na⁺ and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na⁺ pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.     

Ubiquinol-10 and ubiquinone-10 levels in umbilical cord blood of healthy foetuses and the venous blood of their mothers

Abstract

Despite their being good markers of oxidative stress for clinical use, little is known about ubiquinol-10 (reduced coenzyme Q₁₀) and ubiquinone-10 (oxidized coenzyme Q₁₀) levels in foetuses and their mothers. This study investigates oxidative stress in 10 healthy maternal venous, umbilical arterial and venous bloods after vaginal delivery by measuring ubiquinol-10 and ubiquinone-10 levels. Serum ubiquinol-10 and ubiquinone-10 levels were measured by HPLC with a highly sensitive electrochemical detector. Maternal venous ubiquinol-10 and ubiquinone-10 levels were significantly higher than umbilical arterial and venous levels (all p < 0.001). However, the ubiquinone-10/total coenzyme Q₁₀ (CoQ₁₀) ratio, which reflects the redox status, was significantly higher in umbilical arterial and umbilical venous blood compared to maternal venous blood (all p < 0.001). The ubiquinone-10/total CoQ₁₀ ratio was higher in umbilical arterial than in umbilical venous blood (p < 0.01). The present study demonstrated that foetuses were under higher oxidative stress than their mothers.     

Resistance of reaction centers from Rhodobacter sphaeroides against UV-B radiation. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by ultraviolet-B (UV-B, 280–320 nm) radiation have been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides R26. UV-B irradiation results in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm reflecting the formation of the P⁺(Q_AQ_B)^– state. In addition to this effect, the charge recombination accelerates and the damping of the semiquinone oscillation increases in the UV-B irradiated reaction centers. A further effect of UV-B is a 2 fold increase in the half- inhibitory concentration of o-phenanthroline. Some damage to the protein subunits of the RC is also observed as a consequence of UV-B irradiation. This effect is manifested as loss of the L, M and H subunits on Coomassie stained gels, but not accompanied with specific degradation products. The damaging effects of UV-B radiation enhanced in reaction centers where the quinone was semireduced (Q_B ^–) during UV-B irradiation, but decreased in reaction centers which lacked quinone at the Q_B binding site. In comparison with Photosystem II of green plant photosynthesis, the bacterial reaction center shows about 40 times lower sensitivity to UV-B radiation concerning the activity loss and 10 times lower sensitivity concerning the extent of reaction center protein damage. It is concluded that the main effect of UV-B radiation in the purple bacterial reaction center occurs at the Q_AQ_B quinone acceptor complex by decreasing the binding affinity of Q_B and shifting the electron equilibration from Q_AQ_B ^– to Q_A ^–Q_B. The inhibitory effect is likely to be caused by modification of the protein environment around the Q_B binding pocket and mediated by the semiquinone form of Q_B. The UV-resistance of the bacterial reaction center compared to Photosystem II indicates that either the Q_AQ_B acceptor complex, which is present in both types of reaction centers with similar structure and function, is much less susceptible to UV damage in purple bacteria, or, more likely, that Photosystem II contains UV-B targets which are more sensitive than its quinone complex.Abbreviations Bchl bacteriochlorophyll - P Bchl dimer - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - RC reaction center - UV-B ultraviolet-B     

Changes in [C]Atrazine Binding Associated with the Oxidation-Reduction State of the Secondary Quinone Acceptor of Photosystem II

One hypothesis of triazine-type herbicide action in photosynthetic material is that the herbicide molecule competes with a secondary quinone acceptor, B, for a binding site at the reaction center of photosystem II. The binding affinity of B has been suggested to change with its level of reduction, being most strongly bound in its semiquinone form. To test this hypothesis, [¹⁴C]atrazine binding studies have been carried out under different photochemically induced levels of B reduction in Pisum sativum. It is found that herbicide binding is reduced in continuously illuminated samples compared to dark-adapted samples. Decreased binding of atrazine corresponds to an increase in the semiquinone form of B. With flash excitation, the herbicide binding oscillates with a cycle of two, being low on odd-numbered flashes when the amount of semiquinone form of B is greatest. Treatment with NH₂OH was found to significantly decrease the strength of herbicide binding in the dark as well as stop the ability of p-benzoquinone to oxidize the semiquinone form of B. It is suggested that the mode of action of NH₂OH is disruption of quinones or their environment on both the oxidizing and reducing sides of photosystem II. Herbicide binding was found to be unaltered under conditions when p-benzosemiquinone oxidation of the reduced primary acceptor, Q⁻, is herbicide insensitive; weak herbicide binding cannot explain this herbicide insensitivity. It is concluded that the quinone-herbicide competition theory of herbicide action is correct. Also, since quinones are lipophilic the importance of the lipid composition of the thylakoid membrane in herbicide interactions is stressed.     

Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase.

The electron transfer from ubiquinol-2 to ferricytochrome c mediated by ubiquinol:cytochrome c oxidoreductase [E.C. 1.10.2.2] purified from beef heart mitochondria, which contained one equivalent of ubiquinone-10 (Q10), was investigated under initial steady-state conditions. The Q10-depleted enzyme was as active as the Q10-containing one. Double reciprocal plots for the initial steady-state rate versus one of the two substrates at various fixed levels of the other substrate gave parallel straight lines in the absence of any product. Intersecting straight lines were obtained in the presence of a constant level of one of the products, ferrocytochrome c. The other product, ubiquinone-2, did not show any significant effect on the enzymic reaction. Ferrocytochrome c non-competitively inhibited the enzymic reaction against either ubiquinol-2 or ferricytochrome c. These results indicate a Hexa-Uni ping-pong mechanism with one ubiquinol-2 and two ferricytochrome c molecules as the substrates, which involves the irreversible release of ubiquinone-2 as the first product and the irreversible isomerization between the release of the first ferrocytochrome c and the binding of the second ferricytochrome c. Considering the cyclic electron transfer reaction mechanism, this scheme suggests that the binding of quinone or quinol to the enzyme and electron transfer between the iron-sulfur center and cytochrome c1 are rigorously controlled by the electron distribution within the enzyme.     

Subunit component and their roles in the sodium-transport NADH: quinone reductase of a marine bacterium,Vibrio alginolyticus

The sodium-transport respiratory chain NADH: quinone reductase of a marine bacterium, Vibrio alginolyticus, was purified by high-performance liquid chromatography. The purified quinone reductase, which catalyses the reduction of ubiquinone to ubiquinol, was composed of three subunits, α, β and γ, with apparent molecular weights of 52 000, 46 000 and 32 000, respectively. The subunit β contained one molecule of FAD per molecule and catalysed the reduction of ubiquinone to ubisemiquinone. The subunit α contained FMN as a prosthetic group. The quinone reductase was reconstituted from α and βγ, but not from α and β, and the maximum activity was obtained at the equimolar amounts of FAD(β) and FMN(α). The molecular weight of quinone reductase complex was estimated to be 254 000, which corresponded to a dimer of αβγ complex or α₂β₂γ₂. The subunit γ increased the affinity of β for ubiquinone-1. The reaction catalysed by FMN-containing α-subunit was essential for the generation of membrane potential in proteoliposomes and the coupling site of sodium pump in the quinone reductase was localised to this reaction step.     

Electron sweep across four b-hemes of cytochrome bc1 revealed by unusual paramagnetic properties of the Qi semiquinone intermediate

Dimeric cytochromes bc are central components of photosynthetic and respiratory electron transport chains. In their catalytic core, four hemes b connect four quinone (Q) binding sites. Two of these sites, Q_i sites, reduce quinone to quinol (QH₂) in a step-wise reaction, involving a stable semiquinone intermediate (SQ_i). However, the interaction of the SQ_i with the adjacent hemes remains largely unexplored. Here, by revealing the existence of two populations of SQ_i differing in paramagnetic relaxation, we present a new mechanistic insight into this interaction. Benefiting from a clear separation of these SQ_i species in mutants with a changed redox midpoint potential of hemes b, we identified that the fast-relaxing SQ_i (SQ_iF) corresponds to the form magnetically coupled with the oxidized heme b_H (the heme b adjacent to the Q_i site), while the slow-relaxing SQ_i (SQ_iS) reflects the form present alongside the reduced (and diamagnetic) heme b_H. This so far unreported SQ_iF calls for a reinvestigation of the thermodynamic properties of SQ_i and the Q_i site. The existence of SQ_iF in the native enzyme reveals a possibility of an extended electron equilibration within the dimer, involving all four hemes b and both Q_i sites. This substantiates the predicted earlier electron transfer acting to sweep the b-chain of reduced hemes b to diminish generation of reactive oxygen species by cytochrome bc₁. In analogy to the Q_i site, we anticipate that the quinone binding sites in other enzymes may contain yet undetected semiquinones which interact magnetically with oxidized hemes upon progress of catalytic reactions.     

Binding and release kinetics of inhibitors of Q−A oxidation in thylakoid membranes

Oxygen flash yield patterns of dark adapted thylakoid membranes as measured with a Joliot-type O₂-electrode indicate that inhibitors that block the oxidation of the reduced primary quinone Q^?_A of Photosystem II vary greatly in the rate of binding to and release from the inhibitor / Q_B binding environment. The ‘classical’ Photosystem-II herbicides like diuron and atrazine exhibit slow binding and release kinetics, whereas, for example, phenolic inhibitors, o-phenanthroline and synthetic quinones are exchanging quite rapidly with Q_B (about once per second or faster at inhibitor concentrations causing about 50% inhibition of O₂ evolution). No general relationship between the efficiency of the inhibitor and the exchange rate is observed; it depends mainly on the type of inhibitor. Based on the classical Kok model, equations are derived in order to calculate oxygen yields evolved by thylakoids in single-turnover flashes as a function of the rate constants of inhibitor binding to and release from the inhibitor / Q_B binding environment in the presence of an oxidized or semireduced Q_A · Q_B or Q_A · inhibitor complex. Fitting of theoretical and experimental values yields that o-phenanthroline binds much faster to an oxidized than to a semireduced Q_A · Q_B complex. This fits very well with the hypothesis that the Q^?_B affinity to the site is much higher than that of Q_B. In the case of i-dinoseb, however, inhibitor / quinone exchange seems to occur mainly in the semiquinone state. Possibilities to explain this result are discussed.     

The Q-cycle reviewed: How well does a monomeric mechanism of the bc1 complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc₁ complex is reviewed. The data strongly support a mechanism in which the Q_o-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron–sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe–2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q_o-site, and the reduced iron–sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c₁ and liberate the H⁺. When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O₂ is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b_L to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b_L to enhance the rate constant. The acceptor reactions at the Q_i-site are still controversial, but likely involve a “two-electron gate” in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b₁₅₀ phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed.The mechanism discussed is applicable to a monomeric bc₁ complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b_L hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.     

How rapid are the internal reactions of the ubiquinol:cytochrome c 2 oxidoreductase?

The temperature dependence of the partial reactions leading to turn-over of the UQH₂:cyt c ₂ oxidoreductase of Rhodobacter sphaeroides have been studied. The redox properties of the cytochrome components show a weak temperature dependence over the range 280–330 K, with coefficients of about 1 m V per degree; our results suggest that the other components show similar dependencies, so that no significant change in the gradient of standard free-energy between components occurs over this temperature range. The rates of the reactions of the high potential chain (the Rieske iron sulfur center, cytochromes c ₁ and c ₂, reaction center primary donor) show a weak temperature dependence, indicating an activation energy < 8 kJ per mole for electron transfer in this chain. The oxidation of ubiquinol at the Q_z-site of the complex showed a strong temperature dependence, with an activation energy of about 32 kJ mole^–1. The electron transfer from cytochrome b-566 to cytochrome b-561 was not rate determining at any temperature, and did not contribute to the energy barrier. The activation energy of 32 kJ mole^–1 for quinol oxidation was the same for all states of the quinone pool (fully oxidized, partially reduced, or fully reduced before the flash). We suggest that the activation barrier is in the reaction by which ubiquinol at the catalytic site is oxidized to semiquinone. The most economical scheme for this reaction would have the semiquinone intermediate at the energy level indicated by the activation barrier. We discuss the plausibility of this simple model, and the values for rate constants, stability constant, the redox potentials of the intermediate couples, and the binding constant for the semiquinone, which are pertinent to the mechanism of the ubiquinol oxidizing site.Abbreviations (BChl)₂ P870, primary donor of the photochemical reaction center - b/c ₁ complex ubiquinol: cytochrome c ₂ oxidoreductase - cyt b _H cytochrome b-561 or higher potential cytochrome b - cyt b _L cytochrome b-566, or low potential cytochrome b - cyt c ₁, cyt c ₂, cyt c _t cytochromes c ₁ and c ₂, and total cytochrome c (cyt c ₁ and cyt c ₂) - Fe.S Rieske-type iron sulfur center, Q - QH₂ ubiquinone, ubiquinol - Q_z, Q_zH₂, Q_z ^– ubiquinone, ubiquinol, and semiquinone anion of ubiquinone, bound at quinol oxidizing site - Q_z-site ubiquinol oxidizing site (also called Q_o-(outside) - Q_o (Oxidizing) - Q_P (Positive proton potential) site) - Q_c-site uubiquinone reductase site (also called the Q_i-(inside) - Q_R (Reducing), or - Q_N (Negative proton potential) site) - UHDBT 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazol     

FTIR spectroscopic evidence for the involvement of an acidic residue in quinone binding in cytochrome bd from Escherichia coli

In this work, FTIR difference spectroscopy is used to search for possible binding partners and protonable groups involved in the binding of the quinol to cytochrome bd from Escherichia coli. In addition, the electrochemically induced FTIR difference spectra are compared for preparations of the enzyme isolated from cells grown at different oxygen levels in which the quinone content of the membrane is altered. On this basis, difference signals can be tentatively attributed to the vibrational modes of the different quinones types that are associated with the enzyme depending on growth conditions. Furthermore, vibrational modes due to the redox-dependent reorganization of the protein vary depending on the quinone associated with the isolated enzyme. Of particular interest are the observations that a mode at 1738 cm(-1) is decreased and a mode at 1595 cm(-1) is increased as observed in direct comparison to the data obtained from samples grown anaerobically. These signals indicate a change in the protonation state of an aspartic or glutamic acid. Since these changes are observed when the ubiquinone ratio in the preparation increases, the data provide evidence for the modulation of the binding site by the interacting quinone and the involvement of an acidic group in the binding site. The tentative assignments of the vibrational modes are supported by electrochemically induced FTIR difference spectra of cytochrome bd in the presence of the specific quinone binding site inhibitors heptylhydroxyquinoline-N-oxide (HQNO) or 2-methyl-3-undecylquinolone-4. Whereas HQNO leads to strong shifts in the FTIR redox difference spectrum, 2-methyl-3-undecylquinolone-4 induces a specific shift of a mode at 1635 cm(-1), which likely originates from the displacement of the C=O group of the bound quinone.     

Reduction of ubiquinone-1 by ascorbic acid is a catalytic and reversible process controlled by the concentration of molecular oxygen

SUMMARY

To address whether reduction by vitamin C may contribute to the in vivo maintenance of coenzyme Q in the reduced form, we studied the reduction of ubiquinone-1 by ascorbate at pH 7.4. Addition of ascorbate to ubiquinone-1 resulted in rapid O₂ consumption and an increase in the steady-state concentration of ascorbyl radical. The initial rate of O₂ consumption was proportional to the product of [ubiquinone-1] and [ascorbate] whereas [ascorbyl radical] was proportional to the square root of this parameter; both dependencies were in quantitative agreement with each other. The extent of O₂ consumption greatly exceeded the amounts of ubiquinone-1 initially present. Formation of ubiquinol-1 from ubiquinone-1 by ascorbate was reversible, moderate under aerobic conditions, but substantial in the absence or near absence of oxygen. At high O₂ concentration, ascorbate promoted the oxidation of ubiquinol-1 to ubiquinone-1. Addition of sodium dodecyl sulphate dramatically decreased the rate of reaction between ubiquinone-1 and ascorbate, most likely as a result of phase separation of the reagents. A preliminary reaction scheme with putative rate constants for the relevant reactions is presented that quantitatively describes the kinetic behaviour of the process studied. The key reactions in the scheme are electron transfer from ascorbate to ubiquinone-1 with formation of the ascorbyl and ubisemiquinone radical. The reaction of the latter with O₂ is postulated to be responsible for O₂ consumption, with ubiquinone-1 acting as a catalyst. Together, the results demonstrate that the extent of reduction of ubiquinone-1 by ascorbate was controlled by the O₂ concentration and the physical availability of the reactants. As the O₂ concentration in human blood is relatively high and ubiquinone-10 is located exclusively within the lipid phase of lipoproteins where negatively charged ascorbate has little access, our results suggest that direct reduction by ascorbate is unlikely to be responsible for the high reduction percentage observed for plasma coenzyme Q.     

Photoreductant-induced oxidation of Fe2+ in the electron-acceptor complex of Photosystem II

The ferrous ion associated with the electron acceptors in Photosystem II can be oxidized by the unstable semiquinone form of certain high-potential quinones (phenyl-p-benzoquinone, dimethylbenzoquinone and benzoquinone) which are used as electron acceptors. In a flash sequence, alternating oxidation of the iron by the photoreduced semiquinone on odd-numbered flashes is followed by photoreduction of the iron on even-numbered flashes. These reactions are detected by monitoring EPR signals arising from Fe³⁺. The oxidation of the iron can also occur in the frozen state (−30°C) indicating that the high-potential quinone can occupy the Q_B site. The reaction also takes place when the exogenous quinone is added in the dark to samples in which Q_B is already in the semiquinone form. The inhibitors of electron transfer between Q_A⁻ and Q_B, DCMU and sodium formate, block the photoreductant-induced iron oxidation. It is suggested that the iron oxidation takes place through the Q_B site. This unexpected photochemistry occurs under experimental conditions routinely used in studies of Photosystem II. Some previously reported phenomena can be reinterpreted on the basis of these new data.     

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.