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.     

One-electron reduction of D-amino acid oxidase. Kinetics of conversion from the red semiquinone to the blue semiquinone

The reduction of D-amino acid oxidase (DAAO) by hydrated electrons (eaq-) has been studied in the absence and presence of benzoate by pulse radiolysis. The eaq-did not reduce the flavin moiety in DAAO and reacted with the amino acid residues in the protein. In the presence of benzoate, eaq- first reacted with benzoate to yield benzoate anion radical. Subsequently, the benzoate anion radical transferred an electron to the complex of DAAO-benzoate to form the red semiquinone of the enzyme with a second-order rate constant of 1.2 X 10(9) M-1 s-1 at pH 8.3. After the first phase of the reduction, conversion of the red semiquinone to the blue semiquinone was observed in the presence of high concentration of benzoate. This process obeyed first-order kinetics, and the rate increased with an increase of the concentration of benzoate. In addition, the rate was found to be identical with that of the formation of the complex between benzoate and the red semiquinone of DAAO as measured by a stopped-flow method. This suggests that bound benzoate dissociates after the reduction of the benzoate-DAAO complex by benzoate anion radical and that free benzoate subsequently recombines with the red semiquinone of the enzyme to form the blue semiquinone.     

A new species of bound ubisemiquinone anion in QH2: cytochrome c oxidoreductase

Using a combination of EPR and low temperature diffuse reflectance spectroscopy, a new species of semiquinone anion has been detected in QH2:cytochrome c oxidoreductase in submitochondrial particles under conditions of oxidant-induced extra reduction of cytochrome b. In contrast to the previously detected semiquinone anion, this new species is insensitive to antimycin but sensitive to treatment with 2,3-dimercaptopropanol and O2. The two species can easily be distinguished on the basis of their respective EPR properties since they differ in g-value, line width, and microwave power saturation behavior. It is concluded that the two species of semiquinone anion are bound to different domains on QH2:cytochrome c oxidoreductase. The existence of two different semiquinone anions in the enzyme strongly supports a mechanism of electron flow as proposed in the Q-cycle.     

Exploring by pulsed EPR the electronic structure of ubisemiquinone bound at the QH site of cytochrome bo3 from Escherichia coli with in vivo 13C-labeled methyl and methoxy substituents

The cytochrome bo(3) ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O(2) to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo(3) as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 (13)C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of l-methionine with the side chain methyl group (13)C-labeled. The (13)C-labeled quinone isolated from cytochrome bo(3) was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity.     

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers: the response under conditions of multiple reaction-center turnovers

The characteristics of the photocurrent response activated by continuous illumination of planar bilayer membranes containing bacterial reaction centers have been resolved by voltage clamp methods. The photocurrent response to a long light pulse consists of an initial spike arising from the fast, quasi-synchronous electron transfer from the reaction center bacteriochlorophyll dimer, BChl2, to the primary quinone QA. This is followed by a slow relaxation of the current to that promoted by secondary, asynchronous multiple electron transfers from the reduced cytochrome c through the reaction centers to the ubiquinone-10 pool. Currents derived from cytochrome c oxidation that occurs when cytochrome c is associated with the reaction center or when limited by diffusional interaction from solution are recognized. Changes of the ionic strength and pH in the aqueous phase, and the clamped membrane potential (+/- 150 mV), affect the electron-transfer rate between cytochrome c and BChl2. In contrast, the primary light-induced charge separation between BChl2 and QA, or electron transfer between QA on the ubiquinone pool are unaffected. During illumination of reaction center membranes supplemented with cytochrome c and a ubiquinone pool, there is a small but significant steady-state current which is considered to be caused by the re-oxidation of photoreduced quinone by molecular oxygen. In the dark, after illumination of reaction centers supplemented with cytochrome c and a ubiquinone pool, there is a small amount of reverse current resulting from the movement of charges back across the membrane. This reverse current is observed maximally after 400 ms illumination while prolonged illumination diminishes the effect. The source of this current is uncertain, but it is considered to be due to the flux of anionic semiquinone within the membrane profile; this may also be the species that interacts with oxygen giving rise to the steady-state current. It is postulated that when the reaction centers are contained in an alkane-containing phospholipid membrane, in contrast to the in vivo situation, the semiquinone anion formed in the QB site is not tightly bound to the site and can, by exchange-diffusion with the membrane-quinone pool, move away from the site and accumulate in the membrane. However, in the absence, more quantitative work superoxide anion, resulting from O2 interaction with semiquinone of QA, QB or pool cannot be excluded.     

Production of superoxide anion during the oxidation of hemoglobin by menadione.

Menadione in the presence of oxyhemoglobin will accelerate the formation of methemoglobin and result in the generation of superoxide anion. Menadione appears to oxidize slowly ferrohemoglobin to ferrihemoglobin, while forming menadione semiquinone in the process. Menadione semiquinone is known to react with molecular oxygen to yield superoxide anion. The superoxide anion appears to be the source of hydrogen peroxide which accounts for most of the observed methemoglobin formation when hemoglobin is reacted with menadione.     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

The tiron free radical as a sensitive indicator of chloroplastic photoautoxidation.

Wheat chloroplasts photochemically reduced molecular oxygen, as a Hill oxidant in the Mehler reaction, to superoxide anion which then oxidized added 1,2-dihydroxybenzene-3,5-disulfonate to its semiquinone, a comparatively stable free radical at pH 7. The last mentioned reaction was rapid in aqueous solution, but the rate of formation of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone by the chloroplast system was calculated as T1 of 0.6 s. The Mehler reaction, or more specifically the univalent reduction of oxygen by Photosystem I, was rate-limiting so that the 1,2-dihydroxybenzene-3,5-disulfonate seniquinone was a useful spin probe for superoxide anion production at room temperature. The ESR signal of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was proportional to its steady state concentration and decayed in the dark with a T1/2 of 5-6 s. This oxygen-dependent signal was enhanced by mediation of chloroplastic oxygen reduction through methyl viologen. The superoxide anion scavengers ascorbate and L-epinephrine competitively obscured 1,2-dihydroxybenzene-3,5-disulfonate semiquinone formation, butadded superoxide dismutase was not as effective in this role. Partial inhibition by superoxide dismutase was achieved only by preincubation of Photosystem I enriched particles with ten times the endogenous concentration of superoxide dismutase. This and the persistence of a small amount of a 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) oxidizing species in the dark supports the concept of Tiron accessibility but not the superoxide dismutase accessibility of superoxide anion bound in its formative enzyme complex. Benzoquinone and naphthoquinone disulfonate also reacted with superoxide anion, and supported both the Hill reaction and the Mehler reaction as final oxidants of both water and superoxide anion.     

Orientation of the tyrosyl D, pheophytin anion, and semiquinone Q(A)(*)(-) radicals in photosystem II determined by high-field electron paramagnetic resonance

The radical forms of two cofactors and an amino acid in the photosystem II (PS II) reaction center were studied by using high-field EPR both in frozen solution and in oriented multilayers. Their orientation with respect to the membrane was determined by using one-dimensionally oriented samples. The ring plane of the stable tyrosyl radical, Y(D)(*), makes an angle of 64 degrees +/- 5 degrees with the membrane plane, and the C-O direction is tilted by 72 degrees +/- 5 degrees in the plane of the radical compared to the membrane plane. The semiquinone, Q(A)(*)(-), generated by chemical reduction in samples lacking the non-heme iron, has its ring plane at an angle of 72 degrees +/- 5 degrees to the membrane plane, and the O-O axis is tilted by 21 degrees +/- 5 degrees in the plane of the quinone compared to the membrane plane. This orientation is similar to that of Q(A)(*)(-) in purple bacteria reaction centers except for the tilt angle which is slightly bigger. The pheophytin anion was generated by photoaccumulation under reducing conditions. Its ring plane is almost perpendicular to the membrane with an angle of 70 degrees +/- 5 degrees with respect to the membrane plane. This is very similar to the orientation of the pheophytin in purple bacteria reaction centers. The position of the g tensor with respect to the molecule is tentatively assigned for the anion radical on the basis of this comparison. In this work, the treatment of orientation data from EPR spectroscopy applied to one-dimensionally oriented multilayers is examined in detail, and improvements over previous approaches are given.     

Ca2+ and nucleotide dependent regulation of voltage dependent anion channels in the plasma membrane of guard cells.

Using the patch-clamp technique we discovered that the voltage dependent anion channels in the plasma membrane of guard cells are activated by a rise in cytoplasmic Ca2+ in the presence of nucleotides. Upon activation, these anion channels catalyse anion currents 10-20 times higher than in the inactivated state, thus shifting the plasma membrane from a K+ conducting state to an anion conducting state. Prolonged stimulation by depolarizing voltages results in the inactivation of the anion current (t1/2 = 10-12 s). We suggest that activation of the anion channel by Ca2+ and nucleotides is a key event in the regulation of salt efflux from guard cells during stomatal closure.     

Sigmatropic reactions of the aziridinyl semiquinone species. Why aziridinyl benzoquinones are metabolically more stable than aziridinyl indoloquinones

Described herein is the chemistry of aziridinyl semiquinone species, which are formed upon one-electron metabolic reduction of aziridinyl quinone antitumor agents. The semiquinone species undergo a type of electrocyclic reaction known as a 1,5-sigmatropic shift of hydrogen. This reaction converts the aziridinyl group to both ethylamino and amino groups resulting in a loss of cytotoxicity. Since the radical anion conjugate base does not undergo ring opening as fast as the semiquinone, it was possible to determine the semiquinone pK(a) values by plotting the percent sigmatropic products versus pH. Aziridinyl quinones based on benzoquinones, such as DZQ and AZQ, possess semiquinone pK(a) values below neutrality. In contrast, an indole-based aziridinyl quinone possesses a semiquinone pK(a) value of 9.3. Single electron reduction of DZQ and AZQ by NADPH: cytochrome P-450 reductase at physiological pH therefore affords the radical anion without any sigmatropic rearrangement products. In contrast, the same reduction of an aziridinyl indoloquinone affords the semiquinone which is rapidly converted to sigmatropic rearrangement products. These findings suggest that aziridinyl quinone antitumor agents based on indoles will be rapidly inactivated by one electron-reductive metabolism. A noteworthy example is the indoloquinone agent EO9, which is rapidly metabolized in vivo. In contrast, benzoquinone-based aziridinyl quinone antitumor agents such as AZQ, DZQ, and the new benzoquinone analogue RH1 do not suffer from this problem.     

Reactions of Adriamycin with haemoglobin. Superoxide dismutase indirectly inhibits reactions of the Adriamycin semiquinone.

The Adriamycin semiquinone produced by the reaction of xanthine oxidase and xanthine with Adriamycin has been shown to reduce both methaemoglobin and cytochrome c. In air, but not N2, both reactions were inhibited by superoxide dismutase. With cytochrome c, superoxide formed by the rapid reaction of the semiquinone with O2, was responsible for the reduction. However, even in air, methaemoglobin was reduced directly by the Adriamycin semiquinone. Superoxide dismutase inhibited this reaction by removing superoxide and hence the semiquinone by displacing the equilibrium: Semiquinone + O2 in equilibrium or formed from quinone + O2-. to the right. This ability to inhibit indirectly reactions of the semiquinone could have wider implications for the protection given by superoxide dismutase against the cytotoxicity of Adriamycin. Oxidation of haemoglobin by Adriamycin has been shown to be initiated by a reversible reaction between the drug and oxyhaemoglobin, producing methaemoglobin and the Adriamycin semiquinone. Reaction of the semiquinone with O2 gives superoxide and H2O2, which can also react with haemoglobin. Catalase, by preventing this reaction of H2O2, inhibits oxidation of oxyhaemoglobin. Superoxide dismutase, however, accelerates oxidation, by inhibiting the reaction of the semiquinone with methaemoglobin by the mechanism described above. Although superoxide dismutase has a detrimental effect on haemoglobin oxidation, it may protect the red cell against more damaging reactions of the Adriamycin semiquinone.     

Conformation-driven and semiquinone-gated proton-pump mechanism in the NADH-ubiquinone oxidoreductase (complex I)

A novel mechanism for proton/electron transfer is proposed for NADH-quinone oxidoreductase (complex I) based on the following findings: (1) EPR signals of the protein-bound fast-relaxing semiquinone anion radicals (abbreviated as Q(Nf)-) are observable only in the presence of proton-transmembrane electrochemical potential; (2) Iron-sulfur cluster N2 and Q(Nf)- are directly spin-coupled; and (3) The projection of the interspin vector extends only 5A along the membrane normal [Yano, T., Dunham, W.R. and Ohnishi, T. (2005) Biochemistry, 44, 1744-1754]. We propose that the proton pump is operated by redox-driven conformational changes of the quinone binding protein. In the input state, semiquinone is reduced to quinol, acquiring two protons from the N (matrix) side of the mitochondrial inner membrane and an electron from the low potential (NADH) side of the respiratory chain. A conformational change brings the protons into position for release at the P (inter-membrane space) side of the membrane via a proton-well. Concomitantly, an electron is donated to the quinone pool at the high potential side of the coupling site. The system then returns to the original state to repeat the cycle. This hypothesis provides a useful frame work for further investigation of the mechanism of proton translocation in complex I.     

Identification of the reduced primary electron acceptor of Photosystem II as a bound semiquinone anion

The complete absorption difference spectrum of the primary electron acceptor of Photosystem II has been measured at room temperature in subchloroplast fragments prepared with deoxycholate. The shape and amplitude of the spectrum indicate that the primary reaction involves the reduction of one bound plastoquinone molecule per reaction center to its semiquinone anion. In addition two small absorbance band shifts occur near 545 (C550) and 685 nm, which may be due to an influence of the semiquinone on the absorption spectrum of a reaction center pigment.     

The oxidation of tiron by superoxide anion. Kinetics of the reaction in aqueous solution in chloroplasts.

The rate of reaction between superoxide anion (O2) and 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron) was measured with pulse radiolysis-generated O2. A kinetic spectrophotometric method utilizing competition between p-benzoquinone and tiron for O2 was employed. In this system, the known rate of reduction of p-benzoquinone was compared with the rate of oxidation of tiron to the semiquinone. From the concentration dependence of the rate of tiron oxidation, the absolute second order rate constant for the reaction was determined to be 5x10-8 M-minus1-s-minus1. Ascorbate reduced O2 to hydrogen peroxide with a rate constant of 10-8 M-minus1-s-minus1 as determined by the same method. The tiron semiquinone may be used as an indicator free radical for the formation of superoxide anion in biological systems because of the rapid rate of oxidation of the catechol by O2 compared to the rate of O2 formation is most enzymatic systems. Tiron oxidation was used to follow the formation of superoxide anion in swollen chloroplasts. The chloroplasts photochemically reduced molecular oxygen which was further reduced to hydrogen peroxide by tiron. Tiron oxidation specifically required O2 since O2 was consumed in the reaction and tiron did not reduce the P700 cation radical or other components of Photosystem I under anaerobic conditions.     

Isolation of ATPase I, the proton pump of chromaffin-granule membranes.

Cellobiose oxidase from the white-rot fungus Sporotrichum pulverulentum has been purified to homogeneity by a new procedure. The carbohydrate and amino acid compositions of the enzyme have been determined. Cellobiose oxidase contains FAD and cytochrome b prosthetic groups. Mr of the enzyme has been estimated at 74400 by sedimentation equilibrium. The enzyme is a monomer. Optical, fluorescence and e.p.r. spectra of oxidized and reduced cellobiose oxidase have been determined. A preliminary investigation of the substrate specificity of cellobiose oxidase reveals that disaccharides and even some insoluble polysaccharides are substrates, but not monosaccharides. Strong substrate inhibition is seen at high concentrations of cellobiose. This effect is particularly marked when oxygen is the electron acceptor. Cellobiose oxidase is unusual among flavoproteins, since it stabilizes the red anionic flavin semiquinone and forms a sulphite adduct, yet appears to produce the superoxide anion as its primary reduced oxygen product.     

Intramolecular electron transfer in proteins. Radiolysis study of the reductive activation of daunorubicin complexed in egg white apo-riboflavin binding protein

Daunorubicin, an anthracycline antitumor antibiotic, can be complexed in egg white apo-riboflavin binding protein. The reduction of this complex was studied by gamma-radiolysis and pulse radiolysis using COO.- free radicals as reductants. The final products are 7-deoxydaunomycinone intercalated in the protein and thiol groups coming from the reduction of disulfide bonds of the protein, in the respective proportions of 90% and 10%. One-electron reduction of the complex gives daunorubicin semiquinone radical and a disulfide anion. The rate constants of the reactions of COO.- ions with the complex and with the disulfide bond in the protein alone are respectively equal to 2.4 x 10(8) mol-1.L.s-1 and 6.4 x 10(7) mol-1.L.s-1. Daunorubicin semiquinone decays by a first-order process, the rate constant of which is independent of the initial protein and radical concentrations. Without protein, daunorubicin semiquinone undergoes a disproportionation-comproportionation equilibrium [Houée-Levin, C., Gardès-Albert, M., Ferradini, C., Faraggi, M., & Klapper, M. (1985) FEBS Lett. 179, 46-50]. We propose that a protein residue reduces semiquinone by an intramolecular path. This creates an electron hole in the protein which may alter its function. This reduction process is very different from the reduction mechanism of riboflavin binding protein by the same reductant [Faraggi, M., Steiner, J.P., & Klapper, M.H. (1985) Biochemistry 24, 3273-3279]. These results suggest a new deleterious pathway to explain the antitumor and/or cytotoxic effect of this drug.     

Immobilized radicals. IV. Biological semiquinone anions and neutral semiquinones

The semiquinone anion and neutral semiquinone radicals of benzoquinone, vitamin K-1, ubiquinone and plastoquinone were generated in both protic and aprotic solvents and frozen to produce immobilized spectra. The linewidths of the neutral semiquinones were always much larger than those of the corresponding anion radicals. Furthermore, the spectra of the neutral radicals often exhibit fine structure. When compared with in vivo spectra of semiquinones, these model systems suggest that the ubisemiquinone anion radical observed in photosynthetic bacteria can exist in either a protic or aprotic environment. There is also the implication that Signal II in chloroplasts may be a plastosemiquinone radical with a spin distribution similar to that of the neutral radical.     

Determination of glucose oxidase oxidation-reduction potentials and the oxygen reactivity of fully reduced and semiquinoid forms.

The oxidation-reduction potential values for the two electron transfers to glucose oxidase were obtained at pH 5.3, where the neutral radical is the stable form, and at pH 9.3, where the anion radical is the stable form. The midpoint potentials at 25 degrees were: pH 5.3 EFl1ox + e- H+ equilibrium EFlH. Em1 = -0.063 +/- 0.011 V EFlH. + e- + H+ equilibrium EFlredH2 Em2 = -0.065 +/- 0.007 V pH 9.3 EFlox + e- EFi- Em1 = -0.200 +/- 0.010 V EFi- + e- + H+ equilibrium EFlredH- Em2 = -0.240 +/- 0.005 V All potentials were measured versus the standard hydrogen electrode (SHE). The potentials indicated that glucose oxidase radicals are stabilized by kinetic factors and not by thermodynamic energy barriers. The pK for the glucose oxidase radical was 7.28 from dead time stopped flow measurements and the extinction coefficient of the neutral semiquinone was 4140 M-1 cm-1 at 570 nm. Both radical forms reacted with oxygen in a second order fashion. The rate at 25 degrees for the neutral semiquinone was 1.4 X 10(4) M-1 s-1; that for the anion radical was 3.5 X 10(4) M-1 s-1. The rate of oxidation of the neutral radical changed by a factor of 9 for a temperature difference of 22 degrees. For the anion radical, the oxidation rate changed by a factor of 6 for a 22 degrees change in temperature. We studied the oxygen reactivity of the 2-electron reduced form of the enzyme over a wide wavelength range and failed to detect either oxygenated flavin derivatives or semiquinoid forms as intermediates. The rate of reoxidation of fully reduced glucose oxidase at pH 9.3 was dependent on ionic strength.     

Proton translocation by the cytochrome bc1 complexes of phototrophic bacteria: introducing the activated Q-cycle.

The cytochrome bc1 complexes are proton-translocating, dimeric membrane ubiquinol:cytochrome c oxidoreductases that serve as "hubs" in the vast majority of electron transfer chains. After each ubiquinol molecule is oxidized in the catalytic center P at the positively charged membrane side, the two liberated electrons head out, according to the Mitchell's Q-cycle mechanism, to different acceptors. One is taken by the [2Fe-2S] iron-sulfur Rieske protein to be passed further to cytochrome c1. The other electron goes across the membrane, via the low- and high-potential hemes of cytochrome b, to another ubiquinone-binding site N at the opposite membrane side. It has been assumed that two ubiquinol molecules have to be oxidized by center P to yield first a semiquinone in center N and then to reduce this semiquinone to ubiquinol. This review is focused on the operation of cytochrome bc1 complexes in phototrophic purple bacteria. Their membranes provide a unique system where the generation of membrane voltage by light-driven, energy-converting enzymes can be traced via spectral shifts of native carotenoids and correlated with the electron and proton transfer reactions. An "activated Q-cycle" is proposed as a novel mechanism that is consistent with the available experimental data on the electron/proton coupling. Under physiological conditions, the dimeric cytochrome bc1 complex is suggested to be continually primed by prompt oxidation of membrane ubiquinol via center N yielding a bound semiquinone in this center and a reduced, high-potential heme b in the other monomer of the enzyme. Then the oxidation of each ubiquinol molecule in center P is followed by ubiquinol formation in center N, proton translocation and generation of membrane voltage.