B. Andrea Melandri

Mosaic protonic coupling hypothesis for free energy transduction

*B.C.P. Jansen Instituut, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands and Istituto di Chimica Biologica, Universita di Padova, Via F. Marzolo 3, 35100 Padova, +Istituto ed Orto Botanico, Universita di Bologna, Via Irnerio 42, 40126 Bologna, ?Dipartimento di Fisica, Universita della Calabria, 87036 Cosenza, **C.N.R. Unit for Physiology of Mitochondria, Universita di Padova, Via Loredan 16, 35100 Padova, Italy and ++Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, Wales

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 Ehs. 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 Ehs, 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.

The adaptation of the electron transfer chain of Rhodopseudomonas capsulata to different light intensities

(1) Cells of Rhodopseudomonas capsulata (wild-type) were grown photoheterotrophically in a turbidostat under very high and very low light intensity. Membranes were isolated from cells adapted to the respective light conditions and fractionated by sucrose density centrifugation. The molar ratios of ubiquinone and cytochromes c2, c1, b-561 and b-566 per reaction center were 3-fold to 5-fold higher in high-light than in low-light membranes. (2) Most of the Cyt(c1 + c2) and Cyt b-561 detected in dark redox titrations undergoes light-induced redox changes, both in high- and in low-light membranes. (3) The fractions of the total photooxidizable reaction center and Cyt(c1 + c2) oxidized under continuous light in the absence of antimycin are higher in membranes from low-light- than from high-light-grown cells. (4) From these data and results of kinetic studies it is proposed that cyclic electron flow under saturating light intensities is faster in high-light-grown cells.

Calibration of the response of 9-amino acridine fluorescence to transmembrane pH differences in bacterial chromatophores

The spectral characteristics of absorption and fluorescence emission of 9-amino acridine are not altered by the interaction with bacterial chromatophores, except for the attenuation of both the absorption and emission following the formation of a protonic gradient. The lifetime of fluorescence of the dye is significantly affected in the presence of membranes, and even more following illumination. The shortening of the lifetime induced by light is reversible and prevented by nigericin and K+. The onset kinetics of the fluorescence quenching following the generation of an artificial transmembrane pH difference is temperature dependent, with an activation energy of 17 +/- 3 kcal/mol. The effect of pH on the rate constants is consistent with a model assuming that the diffusion of the unprotonated species is the limiting step in the quenching phenomenon. The response of 9-amino acridine to artificially imposed delta pHs has been utilized as a calibration method for the measurements of the light-induced protonic gradient. The apparent inner volume of chromatophores, evaluated from the extraplation of the response at delta pH = 0, was found to be much larger (15- to 40-fold) than the true osmotic volume, indicating that most of the dye is bound to the membrane when accumulated into the inner lumen.

Reconstitution of cyclic electron transport and photophosphorylation by incorporation of the reaction center, cytochrome bc1 complex and ATP synthase from Rhodobacter capsulatus into ubiquinone-10/phospholipid vesicles

The photosynthetic reaction center, the ubiquinol-cytochrome c reductase (EC 1.10.2.2) and the ATP synthase (EC 3.6.1.4) were selectively solubilized and isolated from the intracytoplasmic membranes of photoheterotrophically grown Rhodobacter capsulatus with n -octyl β- d -glucopyranoside. The purified enzymes were co-reconstituted in predetermined stoichiometries in ubiquinol-10 phospholipid vesicles by a two-step procedure. In the first step, reaction centers and ubiquinol-cytochrome c reductase were incorporated randomly by detergent dialysis. Cyclic electron transport between these two membrane complexes was selected by including cytochrome c in the interior of the proteoliposomes. In the second step, the ATP synthase molecules were incorporated by detergent dilution into the preformed proteoliposomes from the outer face of the vesicles. Analysis of the light-induced redox changes of the cytochromes and of the bacteriochlorophyll dimer, in combination with the effect of specific inhibitors demonstrated that an electron transport pathway closely resembling that of the physiological system was reconstructed in the proteoliposomes. Light-driven ATP synthesis was detected at a maximal rate of 36 nmol ATP/min per mg ATP synthase. Variation in the stoichiometry of the protein components indicated that the rate-limiting reaction was ATP synthesis.

The effect of the size of the quinone pool on the electrogenic reactions in the ubiquinol-cytochrome c2 oxidoreductase of Rhodobacter capsulatus. Pool behaviour at the quinone reductase site

(1) Electron transfer within the ubiquinol-cytochrome c2 oxidoreductase (bc1) complex produces a charge separation across the membrane which, in some bacterial chromatophores, can be monitored by the electrochromic band shift of carotenoids. The electrogenic steps are believed to coincide with the electron transfer through the cytochrome b chain to the quinone reductase site (Qc) of the complex. (2) The kinetics of these electrogenic reactions have been studied in chromatophores from Rhodobacter capsulatus, activated by a single turnover flash, as a function of the ambient redox potential, and of the size of the ubiquinone pool in isooctane-extracted membranes. (3) In chromatophores containing the native ubiquinone (UQ) pool the rate of charge transfer, monitored by the electrochromic red shift of caretenoids, is progressively increased by lowering the Eh below 160 mV. At 100 mV the rate, when normalized to the number of reaction centers turning over, reaches a maximum and declines significantly when the Eh is lowered further (down to 40 mV). This rate remains constant for Eh values between 40 mV and −40 mV. (4) When the size of the UQ pool is decreased by isoctane extraction, the stimulation in the rate of charge separation takes place only at more negative Eh values and the value of the maximum rate is decreased. Also at negative Eh values, however, when no pre-oxidized quinone from the pool is present in the membrane, the rate does not decline significantly from its maximum value. (5) The size of the UQ pool affects also the number of charges translocated per flash by the bc1 complex, since it affects the probability of rapid multiple turnover of the complex, which occurs at Eh < 160 mV in unextracted membranes. (6) These data are interpreted as evidence that oxidized UQ from the pool interacts collisionally with the Qc site. At Eh < 40 mV, when the pool is totally reduced before the flash, the oxidized UQ molecule produced at every turnover at the Qz site of the bc1 complex is sufficient for sustaining a rather high reaction rate at the Qc site. The apparent Km estimated for quinone reduction at the Qc site is approx. 1 mM, less than one order of magnitude smaller than that estimated for QH2 at the Qz site. The rate of transfer of ubiquinone from Qz to Qc required by such a Q cycle mechanism does not appear incompatible with the experimental data.

Intrinsic uncoupling in the ATP synthase of Escherichia coli

The ATP hydrolysis activity and proton pumping of the ATP synthase of Escherichia coli in isolated native membranes have been measured and compared as a function of ADP and Pi concentration. The ATP hydrolysis activity was inhibited by Pi with an half-maximal effect at 140 microM, which increased progressively up in the millimolar range when the ADP concentration was progressively decreased by increasing amounts of an ADP trap. In addition, the relative extent of this inhibition decreased with decreasing ADP. The half-maximal inhibition by ADP was found in the submicromolar range, and the extent of inhibition was enhanced by the presence of Pi. The parallel measurement of ATP hydrolysis activity and proton pumping indicated that, while the rate of ATP hydrolysis was decreased as a function of either ligand, the rate of proton pumping increased. The latter showed a biphasic response to the concentration of Pi, in which an inhibition followed the initial stimulation. Similarly as previously found for the ATP synthase from Rhodobacter caspulatus [P. Turina, D. Giovannini, F. Gubellini, B.A. Melandri, Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, Biochemistry 43 (2004) 11126-11134], these data indicate that the E. coli ATP synthase can operate at different degrees of energetic coupling between hydrolysis and proton transport, which are modulated by ADP and Pi.

Physiological Ligands ADP and Pi Modulate the Degree of Intrinsic Coupling in the ATP Synthase of the Photosynthetic Bacterium Rhodobacter capsulatus

The proton-pumping and the ATP hydrolysis activities of the ATP synthase of Rhodobacter capsulatus have been compared as a function of the ADP and P(i) concentrations. The proton pumping was measured either with the transmembrane pH difference probe, 9-amino-6-chloro-2-methoxyacridine, or with the transmembrane electric potential difference probe, bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol, obtaining consistent results. The comparison indicates that an intrinsic uncoupling of ATP synthase is induced when the concentration of either ligand is decreased. The half-maximal effect was found in the submicromolar range for ADP and at about 70 microM for P(i). It is proposed that a switch from a partially uncoupled state of ATP synthase to the coupled state is induced by the simultaneous binding of ADP and P(i).

The Stimulation of Photophosphorylation and ATPase by Artificial Redox Mediators in Chromatophores of Rhodopseudomonas capsulata at Different Redox Potentials

Abstract(1) Inhibition of cyclic phosphorylation in chromatophores ofRhodopseudomonas capsulata by antimycin A can be fully reversed by artificial redox mediators, provided the ambient redox potential is maintained around 200 mV. The redox mediator need not be a hydrogen carrier in its reduced form, N-methyl-phenazonium methosulfate and N,N,N′,N′-tetramethyl-p-phenylenediamine being equally effective. However, the mediator needs to be lipophilic. Endogenous cyclic phosphorylation is fastest around 130 mV. A shift to 200 mV can also be observed if high concentrations of artificial redox mediator are present in the absence of antimycin A. (2) ATPase activity ofRhodopseudomonas capsulata, in the light as well as in the dark, activated or not activated by inorganic phosphate, can also be stimulated by N-methylphenazonium methosulfate. This stimulation is highest at redox potentials between 60 to 80 mV and is sensitive to antimycin A. In this case N,N,N′,N′-tetramethyl-p-phenylenediamine is much less effective.

Kinetic measurements of electron transfer in coupled chromatophores from photosynthetic bacteria A method of correction for the electrochromic effects

A quantitative study of the kinetics of electron transfer under coupled conditions in photosynthetic bacteria has so far been prevented by overlap of the electrochromic signals of carotenoids and bacteriochlorophyll with the absorbance changes of cytochromes and reaction centers. In this paper a method is presented by which the electrochromic contribution at any wavelength can be calculated from the electrochromic signal recorded at 505 nm, using a set of empirically determined polynomial functions. The electrochromic contribution to kinetic changes at any wavelength can then be subtracted to leave the true kinetics of the redox changes. The corrected redox changes of the reaction center measured at 542 and 605 nm mutually agree, thus providing an excellent test of self‐consistency of the method. The corrected traces for reaction center and of cytochrome b‐566 demonstrate large effects of the membrane potential on the rate and poise of electron transfer. It will now be possible to study the interrelation between proton gradient and individual electron transfer reactions under flash or steady‐state illumination.