Ubiquinol-cytochrome C2 Oxidoreductase Research Articles

The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase

Atomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization.

Q-band ENDOR spectra of the Rieske protein from Rhodobactor capsulatus ubiquinol-cytochrome c oxidoreductase show two histidines coordinated to the [2Fe-2S] cluster.

Electron nuclear double resonance (ENDOR) experiments were performed on 14N (natural abundance) and 15N-enriched iron-sulfur Rieske protein in the ubiquinol-cytochrome c2 oxidoreductase from Rhodobactor capsulatus. The experiments proved that two distinct nitrogenous ligands, histidines, are undoubtedly ligated to the Rieske [2Fe-2S] center. The calculations of hyperfine tensors give values similar but not identical to those of the Rieske-type cluster in phthalate dioxygenase of Pseudomonas cepacia and suggest a slightly different geometry of the iron-sulfur cluster in the two proteins.

Mutants of ubiquinol-cytochrome c2 oxidoreductase resistant to Qo site inhibitors: consequences for ubiquinone and ubiquinol affinity and catalysis

Seven single-site mutants in six residues of the cyt b polypeptide of Rhodobacter capsulatus selected for resistance to the Qo site inhibitors stigmatellin, myxothiazol, or mucidin [Daldal, F., Tokito, M.K., Davidson, E., & Faham, M. (1989) EMBO J. 8, 3951-3961] have been characterized by using optical and EPR spectroscopy and single-turnover kinetic analysis. The strains were compared with wild-type strain MT1131 and with the Ps- strain R126 (G158D), which is dysfunctional in its Qo site [Robertson, D.E., Davidson, E., Prince, R.C., van den Berg, W.H., Marrs, B.L., & Dutton, P.L. (1986) J. Biol. Chem. 261, 584-591]. Mutants selected for stigmatellin resistance induced a weakening in the binding of the inhibitor without discernible loss of ubiquinone(Q)/ubiquinol(QH2) binding affinity to the Qo site or kinetic impairment to catalysis. Mutants selected for myxothiazol or mucidin resistance, inducing weakening of inhibitor binding, all displayed impaired rates of Qo site catalysis: The most severe cases (F144L, F144S) displayed loss of affinity for Q, and evidence suggests that parallel loss of affinity for the substrate QH2 was incurred in these strains. The results provide a view of the nature of the interaction of Q and QH2 of the Qpool with the Qo site. Consideration of the mutational substitutions and their structural positions along with comparisons with the QA and QB sites of the photosynthetic reaction center suggests a model for the structure of the Qo site.

Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides.

The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.

Cloning and sequencing of the fbcF, B and C genes encoding the cytochrome b/c 1 complex from Rhodopseudomonas viridis

The complete nucleotide sequence of the genes encoding the Rieske FeS, the cytochrome b and the cytochrome c1 subunits of the ubiquinol-cytochrome c2 oxidoreductase from the photosynthetic purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequences are presented. These three genes, fbcF, fbcB and fbcC, are located at contiguous sites of the genome. The DNA-deduced amino acid sequences are compared with known primary structures of corresponding proteins from other purple photosynthetic bacteria, as well as mitochondria, cyanobacteria and chloroplasts.

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 c 2 oxidoreductase ( bc 1) 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 (Q c) 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 E h 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 E h is lowered further (down to 40 mV). This rate remains constant for E h 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 E h values and the value of the maximum rate is decreased. Also at negative E h 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 bc 1 complex, since it affects the probability of rapid multiple turnover of the complex, which occurs at E h < 160 mV in unextracted membranes. (6) These data are interpreted as evidence that oxidized UQ from the pool interacts collisionally with the Q c site. At E h < 40 mV, when the pool is totally reduced before the flash, the oxidized UQ molecule produced at every turnover at the Q z site of the bc 1 complex is sufficient for sustaining a rather high reaction rate at the Q c site. The apparent K m estimated for quinone reduction at the Q c site is approx. 1 mM, less than one order of magnitude smaller than that estimated for QH 2 at the Q z site. The rate of transfer of ubiquinone from Q z to Q c required by such a Q cycle mechanism does not appear incompatible with the experimental data.

The nature and magnitude of the charge-separation reactions of ubiquinol cytochrome c2 oxidoreductase

The transdielectric charge separation reaction catalyzed by the ubiquinol-cytochrome c 2 oxidoreductase is achieved in two fractional steps. We present a detailed analysis which addresses the nature of the charge transferred, the redox groups directly involved in charge separation and the contributions of each to the full charge separation catalyzed by the enzyme. Accounting for light saturation effects, reaction centers unconnected to cytochrome c 2 and the fraction of total cytochrome bc 1 turning over per flash permits detailed quantitation of: (1) the red carotenoid bandshift associated with electron transfer between ubiquinol at site Q z and the high- (2Fe2S center, cytochrome c 1) and low-potential (cytochrome b L, cytochrome b H) components of cytochrome bc t; (2) the blue bandshift accompanying reduction of cytochrome b H by ubiquinol via site Q c (the reverse of the physiological reaction); and (3) the effect of δϕ on the Q c -cytochrome b H redox equilibrium. Studies were performed at pH values above and below the redox-linked p K values of the redox centers known to be involved in each reaction at equilibrium. The conclusions of this study may be summarized as follows: (1) there is no transdielectric charge separation apparent in the redox reactions between Q z and cytochrome b L, 2Fe2S and cytochrome c 1 (in agreement with Glaser, E. and Crofts, A.R. (1984) Biochim. Biophys. Acta 766, 223–235), i.e., charge separation accompanies electron transfer between cytochrome b L and cytochrome b H; (2) the redox reactions between cytochrome b L and cytochrome b H and between cytochrome b H and Q c constitute the full electrogenic span; (3) electron transfer between cytochrome b L and cytochrome b H contributes approx. 60% of this span; (4) electron transfer between cytochrome b H and Q c contributes 45–55% as calculated from the blue bandshift or the Δϕ-dependent equilibrium shift; (5) there is no discernable pH dependence of the Q z-cytochrome b H or Q c -cytochrome b H charge-separation reactions; (6) cytochrome b L, Q z, 2Fe2S, and cytochrome c 1 are on the periplasmic side out of the low dielectric part of the membrane while cytochrome b H is buried in the low dielectric medium; (7) electron transfer is the predominant if not the sole contributor to charge separation; (8) Q z and Q c are on opposite sides of the membrane dielectric profile.

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

(1) 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. (2) 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 E h range between 170 and 100 mV (pH 7), is also stimulated in partially UQ-depleted membranes, although at more negative E h's. When the UQ pool is completely extracted the rate of cytochrome (Cyt) b-561 reduction is low and unaffected by the redox potential. (3) In membranes enriched in UQ-10 above the physiological level the titration curve of the rate of Cyt b-561 reduction is displaced to E h 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. (4) 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 E h 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 E h's, respectively. Similarly, the lag becomes E h independent and markedly longer than in controls when the pool is completely extracted. (5) These results demonstrate that the rate of turnover of the ubiquinol oxidizing site in the b- c 1 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 K m of 3–5 ubiquinol molecules per reaction center.

Discrete catalytic sites for quinone in the ubiquinol-cytochrome c2 oxidoreductase of Rhodopseudomonas capsulata. Evidence from a mutant defective in ubiquinol oxidation.

A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.

Photosynthetic membrane development in Rhodopseudomonas sphaeroides. Spectral and kinetic characterization of redox components of light-driven electron flow in apparent photosynthetic membrane growth initiation sites.

The kinetics of light-driven electron flow and the nature of redox centers at apparent photosynthetic membrane growth initiation sites in Rhodopseudomans sphaeroides were compared to those of intracytoplasmic photosynthetic membranes. In sucrose gradients, these membrane growth sites sediment more slowly than intracytoplasmic membrane-derived chromatophores and form an upper pigmented band. Cytochromes c1, c2, b561, and b566 were demonstrated in the upper fraction by redox potentiometry; c-type cytochromes were also detected electrophoretically. Signals characteristic of light-induced reaction center bacteriochlorophyll triplet and photooxidized reaction center bacteriochlorophyll dimer states were observed by EPR spectroscopy but the Rieske iron-sulfur signal of the ubiquinol-cytochrome c2 oxidoreductase was present at a 3-fold reduced level on a reaction center basis in comparison to chromatophores. Flash-induced absorbance measurements of the upper pigmented fraction demonstrated reaction center primary and secondary semiquinone anion acceptor signals, but cytochrome b561 photoreduction and cytochrome c1/c2 reactions occurred at slow rates. This fraction was enriched approximately 2- and 4-fold in total b- and c-type cytochromes, respectively, per reaction center over chromatophores, but photoreducible b-type cytochrome was lower. Measurements of respiratory activity indicated a 1.6-fold higher level of succinate-cytochrome c oxidoreductase/reaction center than in chromatophores, but the apparent turnover rates in both preparations were low. Overall, the results suggest that complete cycles of rapid, light-driven electron flow do not occur merely by introduction of newly synthesized reaction centers into respiratory membrane, but that subsequent synthesis and assembly of appropriate components of the ubiquinol-cytochrome c2 oxidoreductase is required.

Reduction of cytochrome b-561 through the antimycin-sensitive site of the ubiquinol-cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides

Cytochrome b-561 of the ubiquinol-cytochrome c 2 oxidoreductase complex of Rhodopseudomonas sphaeroides is reduced after flash illumination in the presence of myxothiazol in an antimycin-sensitive reaction. Flash-induced reduction was observed over the redox range in which cytochrome b-561 and the Q-pool are both oxidized before the flash. The extent of reduction increased with increasing pH, and was maximal at pH > 10.0 where the extent approached that observed in the presence of antimycin following a group of flashes. Reduction of cytochrome b-561 in the presence of myxothiazol showed a lag of ~ 1 ms after the flash, followed by reduction with ( t 1 2 ~ 6 ms; by analogy with the similar kinetics of the quinol oxidase site, we suggest that the rate is determined by collision with the QH 2 produced in the pool on flash excitation.

Phospholipid-enriched bacterial chromatophores. A system suited to investigate the ubiquinone-mediated interactions of protein complexes in photosynthetic oxidoreduction processes.

Fusion of phospholipid vesicles with photosynthetic chromatophores from Rhodopseudomonas sphaeroides was induced by freezing and thawing. After sucrose density gradient sedimentation, bands containing closed vesicles characterized by different phospholipid to reaction center molar ratios could be isolated and analyzed morphologically and functionally by means of electron microscopy and fast spectroscopy, respectively. Analogously to data reported for phospholipid-enriched mitochondrial inner membranes (Schneider, H., Lemasters, J. J., and Hackenbrock, C. R. (1982) J. Biol. Chem. 257, 10793), the rate of photosynthetic electron transfer in phospholipid-enriched chromatophores decreased with increasing distance between integral membrane complexes. A fast cyclic electron transfer could be restored when the concentration of the ubiquinone pool within the lipid bilayer was reconstituted by additions of exogenous ubiquinone. These results suggest that cyclic electron transfer between reaction center and ubiquinol-cytochrome c2 oxidoreductase complexes in phospholipid-enriched chromatophores is limited by the lateral diffusion of the quinone molecules in the membrane plane. The observation that dilution of the quinone pool in the lipid bilayer affects the rate of photosynthetic electron transport contrasts with previously reported data which indicated that up to 80% of the quinone pool can be removed without altering the kinetic parameters of the overall process. These conflicting results can be reconciled by a model which assumes that the relative orientation of the protein complexes, possibly controlled by protein-protein interactions within the lipid bilayer, plays a key role in the effectiveness of the molecular collisions. According to a diffusion-limited mechanism, this would lead to a fast electron transfer during the photosynthetic reactions.

Characterization of cytochrome b in the isolated ubiquinol-cytochrome c2 oxidoreductase from Rhodopseudomonas sphaeroides GA

Extinction coefficients for cytochrome b and c 1 in the isolated cytochrome bc 1 complex from Rhodopseudomonas sphaeroides GA have been determined. They are 25 mM −1.cm −1 at 561 nm for cytochrome b and 17.4 mM −1.cm −1 at 553 nM for cytochrome c 1 for the difference between the reduced and the oxidized state. Cytochrome b is present in two forms in the complex. One form has an E m7 of 50 mV, an α-peak of 557 nm at liquid N 2 temperature and of 561 nm at RT, which is red-shifted by antimycin A. The other form has an E m7 of −90 mV, a double α-peak of 555 and 561 nm at liquid N 2 temperature corresponding to 559 and 566 nm at RT. The absorption at 566 nm is red-shifted by myxothiazol. The two shifts are independent of each other. Both midpoint potentials of cytochromes b are pH-dependent. The redox center compositions of the cytochrome bc 1 complexes from Rhodopseudomonas sphaeroides and from mitochondria are identical.