THE ROLE OF THE QUINONE POOL IN THE CYCLIC ELECTRON-TRANSFER CHAIN OF RHODOPSEUDOMONAS SPHAEROIDES: A MODIFIED Q-CYCLE MECHANISM

Abstract

(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c(2) oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c(1) and c(2), the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0-3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c(1) and c(2), and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 is oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c(2) oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c(1) and c(2) is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.

Fig. 1

The extent and initial rate of cytochrome b-561 reduction, and reaction center rereduction, as a function of flash number. Traces A and B represent the reduction (upward deflection) of cytochrome b-561, measured at 561 nm minus 569 nm, after one or two flashes, respectively. Traces C and D, flash one and two respectively, show the effect of flash number on reaction center rereduction, measured at 542 nm. The redox potential was adjusted to 400±2 mV by addition of small amounts of ferricyanide. The redox mediators used were phenazine methosulfate, phenazine ethosulfate, pyocyanin and TMPD at 1 μM; and 1,2-naphthoquinone, 1,4-naphthoquinone and duroquinone at 10 μM. Traces (average of four, 200 μs instrument response time) were obtained from chromatophores (approx. 0.6 μM reaction center) suspended in buffer (50 mM Mops, 100 mM KCI, pH 7.0), and excited by two consecutive flashes 5 s apart.

Fig. 2

Kinetic traces of cytochrome b-561 reduction at different values of E_h. Left column: traces showing the acceleration in rate of reduction after one short flash (sweep 10 ms full scale, time constant 10 μs, average of 16). The two traces on the right, top, show the full extent of cytochrome b-561 reduction after one flash, at 200 and 120 mV, respectively (sweep 100 ms full scale, time constant 100 μs, average of two). The two lower traces are the reduction rate after two flashes places 700 μs apart at two values of E_h (sweep 10 ms full scale, time constant 10 μs, average of 16). The mediators present were 1 μM each of phenazine methosulfate, phenazine ethosulfate and pyocyanin; 10 μM each of 1,2-naphthoquinone, 1,4-naphthoquinone, p-benzoquinone and duroquinone; and 2 μM DAD. Valinomycin and nigericin at 2 μM and antimycin at 10 μM were also added. The reaction center concentration was 0.42 μM for all traces. A dark period of 60 s was allowed between flashes or pairs of flashes.

Fig. 3

Titration of extent and kinetics of reduction of cytochrome b-561. The filled circles show initial rates for cytochrome b-561 reduction from traces such as those in Fig. 2, assuming a value of 20 mM⁻¹·cm⁻¹ at 561–569 nm for the extinction coefficient of cytochrome b-561 and 1 cytochrome b-561 per oxidoreductase complex. In these experiments a flash of duration of approx. 3.5 μs at half amplitude was used. The open circles show the rates at low E_h obtained by using a flash with longer duration (25 μs), for comparison with the results of Figs. 7 and 8 in which a similar flash was used. The differences between the two curves can be ascribed to double hits of the reaction center using the longer flash. The crosses show the titration when two short (3.5 μs) flashes were used with 700 μs between the flashes. The total extents of cytochrome b-561 reduction after one flash are plotted using open diamonds. The lag times between the flash and the start of cytochrome b-561 reduction are shown with full diamonds. RC, reaction center.

Fig. 4

Second-order kinetics of cytochrome b-561 reduction. Kinetic traces of cytochrome b-561 reduction as in Fig. 2 were analyzed for second-order reaction. In this figure, ln([QH₂]/[b-561(ox)]) is plotted against time for several values of E_h. The estimates of initial values for [QH₂] were based on the postulates of the model (see text). The concentration of oxidized cytochrome b-561 was estimated on the assumption that all cytochrome b-561 available for reduction was initially oxidized at these values of E_h. The straight lines show good agreement with second order kinetics. (See text for further assumptions.)

Fig. 5

Kinetic traces of the carotenoid electrochromic change. Chromatophores were suspended in the same buffer and with the same mediators as in Fig. 1 with the exception that TMPD was omitted and 10 μM p-benzoquinone was added. The carotenoid band shift was measured at 503 nm. All traces labelled A were obtained in the absence of inhibitors. Traces labelled B were obtained in the presence of 2 μM antimycin A. Traces labelled C at 250 and 100 mV are the difference between the traces labelled A and B. The horizontal scale bar represents 25 ms (traces at 250, 190 and 150 mV) or 5 ms (130, 120 and 100 mV). The instrument response time was 100 μs for the former and 20 μs for the latter sets. Traces at 130, 120 and 100 mV are an average of two. A dark period of 60s was allowed between flashes.

Fig. 6

Titration of the extent and initial rate of carotenoid electrochromic change. Conditions were the same as described in Fig. 5. The open circles represent the extent of the fast phase (Phases I + II), the closed circles represent the maximal extent of the slow phase (Phase III), and the closed triangles represent the initial rate of the slow phase. The initial rate and maximal extent of the slow phase were measured from the difference between traces without and with antimycin (see traces C in Fig. 5). The scales were derived from the following assumptions. The amplitude of the electrochromic change was considered to the proportional to the change in voltage difference generated across the membrane (see Refs. – for reviews). At fixed capacitance this would be proportional to the quantity of charge displaced. The rate of change of absorbance with time therefore measures the current through the electrogenic process, expressed here in units of electrons/ms. It was further assumed (see text) that the full extent of the slow phase (ΔA = 9.6·10⁻³) represented a flux of two electrons per oxidoreductase complex through the electrogenic process.

Fig. 7

Kinetics of cytochrome c₁ and c₂ in the absence and presence of antimycin A. Traces (average of four; instrument response times were 20, 40 and 100 μs for traces at 105, 150 and 245 mV, respectively) were measured at 551–542 nm, which measures almost equal contributions from cytochrome c₁ and cytochrome c₂. Conditions were the same as in Fig. 5 except that gramicidin at 10 μg/ml was added, and a flow system was used to provide a fresh dark sample for each experiment [38]. The concentration of antimycin was 2μM where indicated. Traces were measured at the E_h shown ± 3 mV.

Fig. 8

Redox titration of the initial rate and extent of the antimycin-sensitive cytochrome c rereduction. Conditions were the same as in Fig. 5. The initial rate and extent were measured from the difference traces, without minus with antimycin, obtained as in Fig. 7. The closed triangles correspond to the initial rate of the antimycin-sensitive rereduction, closed circles represent the maximal extent of rereduction, the open squares represents the extent 25 ms after the flash, and the closed squares the extent 4 ms after the flash.

SCHEME I. THE REACTIONS OF THE Q-CYCLE INVOLVED IN UBIQUINOL OXIDATION

See text for explanation. [${\mathrm{Q}}_{\stackrel{.}{\mathrm{z}}}^{-}$] is the semiquinone assumed to be formed transiently at the catalytic site.

SCHEME II. A MODIFIED Q-CYCLE MECHANISM FOR THE CYCLIC ELECTRON TRANSFER CHAIN OF RPS. SPHAEROIDES

The numbered processes represent the following chemical reactions. $$\mathrm{cyt}{c}_2+{\mathrm{P}}^{+}\rightleftharpoons \mathrm{cyt}{c}_2+\mathrm{P}$$ $$[\mathrm{FeS}\cdot \mathrm{cyt}{c}_1]+\mathrm{cyt}{c}_2^{+}\rightleftharpoons [\mathrm{FeS}\cdot \mathrm{cyt}{c}_1^{+}]+\mathrm{cyt}{c}_2$$ $$[\mathrm{FeS}\cdot \mathrm{cyt}{c}_1^{+}]\rightleftharpoons [{\mathrm{FeS}}^{+}\cdot \mathrm{cyt}{c}_1]$$ $${\mathrm{QH}}_2+[{\mathrm{FeS}}^{+}\cdot \mathrm{cyt}b-566]\rightleftharpoons \mathrm{Q}+[\mathrm{FeS}\cdot \mathrm{cyt}b-566\mathrm{H}]+{\mathrm{H}}_{\mathrm{P}}^{+}$$ $$[\mathrm{cyt}b-566\mathrm{H}\cdot \mathrm{cyt}b-561]\rightleftharpoons [\mathrm{cyt}b-566\cdot \mathrm{cyt}b-{561}^{-}]+{\mathrm{H}}_{\mathrm{P}}^{+}$$ $$\begin{array}{cc}\hfill [\mathrm{cyt}& b-566\mathrm{H}\cdot \mathrm{cyt}b-{}^{-}561]+2{\mathrm{H}}_{\mathrm{N}}^{+}+\mathrm{Q}\hfill \\ \hfill & \rightleftharpoons [\mathrm{cyt}b-566\cdot \mathrm{cyt}b-561]+{\mathrm{QH}}_2+{\mathrm{H}}_{\mathrm{P}}^{+}\hfill \end{array}$$ For complete oxidation of one equivalent of QH₂ and reduction of 2P⁺, reactions 1–4 have to occur twice, and reactions 5 and 6 once. The subscripts N and P indicate the phases with which the protons equilibrate. Q and QH₂ are ubiquinone and ubiquinol in the pool. ${\mathrm{Q}}_{\stackrel{.}{\mathrm{B}}}^{-}$ ,${\mathrm{Q}}_{\stackrel{.}{\mathrm{C}}}^{-}$ and ${\mathrm{Q}}_{\stackrel{.}{\mathrm{z}}}^{-}$, are semiquinone species stabilized (or transiently formed) at the catalytic sites. See text for details.