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J. Biol. Chem., Vol. 275, Issue 34, 26121-26127, August 25, 2000

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Kinetic Effects of the Electrochemical Proton Gradient on Plastoquinone Reduction at the Qi Site of the Cytochrome b₆f Complex^*

Romina Paola Barbagallo^§, Cécile Breyton^¶, and Giovanni Finazzi^**

From the  Centro di Studio del CNR sulla Biologia Cellulare e Molecolare delle Piante, via Celoria 26, 20133 Milano, Italy and the ^¶ Max Planck Institute of Biophysics, Department of Structural Biology, Heinrich-Hoffmann-Strasse 7, D-60528 Frankfurt am Main, Germany

Received for publication, March 20, 2000, and in revised form, June 12, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have investigated the effects of the light-induced thylakoid transmembrane potential on the turnover of the b₆f complex in cells of the unicellular green alga Chlamydomonas reinhardtii. The reduction of the potential by either decreasing the light intensity or by adding increasing concentrations of the ionophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) revealed a marked inhibition of the cytochrome b₆ oxidation rate (10-fold) without substantial modifications of cytochrome f oxidation kinetics. Partial recovery of this inhibition could be obtained in the presence of ionophores provided that the membrane potential was re-established by illumination with a train of actinic flashes fired at a frequency higher than its decay. Measurements of isotopic effects on the kinetics of cytochrome b₆ oxidation revealed a synergy between the effects of ionophores and the H₂O-D₂O exchange. We propose therefore, that protonation events influence the kinetics of cytochrome b₆ oxidation at the Qi site and that these reactions are strongly influenced by the light-dependent generation of a transmembrane potential.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cytochrome b₆f complex is a central component of the photosynthetic chain, transferring electrons from the photosystem II (PSII)¹ to the photosystem I (PSI). It comprises four major subunits (1, 2): the Rieske protein, which binds a Fe₂S₂ cluster, with an E_m of +290 mV (3), a c-type cytochrome, cytochrome f, with an E_m of +330 mV (2), a b-type cytochrome, cytochrome b₆, which binds the high and low potential hemes, b_h and b_l, with E_m values of 84 mV and 158 mV, respectively (2), and the subunit IV. Four additional small subunits PetG, PetL, PetM and PetN (2, 4-5) have also been evidenced. Sequence comparisons have shown that cytochrome b₆ and subunit IV are, respectively, homologous to the N- and C-terminal parts of mitochondrial and bacterial cytochromes b (6).

As a member of the bc-type proteins, cytochrome b₆f complex couples proton translocation across the membrane to electron transfer from a lipophilic two-electron donor (plastoquinol) to a hydrophilic one-electron acceptor protein (plastocyanin or a c-type cytochrome). This electron transfer operates through a high potential chain (also called the linear path) formed by the Rieske protein and cytochrome f. The most widely accepted hypothesis to explain the function mechanism of bc complexes is the "Q cycle" hypothesis of Mitchell (7). It predicts that oxidation of quinols not only involves the linear path but also a cyclic path composed of the b hemes, b_l and b_h, and termed the low potential chain. This mechanism, as modified by Crofts et al. (8), postulates both an oxidation and a reduction of plastoquinol at two distinct sites of the protein, the Qo and Qi sites, on opposite sides of the membrane. The oxidation of plastoquinol at the Qo site is associated with the reduction of both cytochrome f and b_l (7-8) and the release of two protons into the lumen.

The recent elucidation of the structure of the respiratory bc₁ complex, the mitochondrial analogue of the b₆f complex, by x-ray crystallography (9-11) suggests a mechanism for plastoquinol oxidation at the Qo site (9-11). It has been reported that the extramembrane domain of the Rieske protein assumes different conformations with respect to its transmembrane helix (10-13). A movement of the quinol molecule has also been predicted, on the basis of the position within the Qo site of residues responsible for the resistance to different inhibitors (10-13). An interpretative model has been then proposed (11, 14) according to which the quinol and the oxidized Rieske protein remain close together at the Qo site until an electron is transferred from the quinol to the iron of the Rieske. They then move apart to reduce the b_l and the c heme, respectively. We have recently confirmed the validity of this model in the case of the cytochrome b₆f complex, measuring the effects of different inhibitors of the Qo site on the kinetics of electron injection into the low and the high potential paths (15).

Less information is available on the oxidation mechanism of the b₆ hemes (16-18). According to the Q cycle, this occurs through a two-step reduction of a plastoquinone molecule at the Qi site. Thus, under oxidizing conditions, the electron transfer sequence is the reduction by plastoquinol of cytochrome b_l, which is in turn oxidized by cytochrome b_h. A second turnover places both b_l and b_h in a reduced state and causes the reduction of a plastoquinone to a plastoquinol at the Qi site, b hemes reoxidation, and proton uptake from the stromal space. The redox cofactors involved in plastoquinol oxidation through the linear path (the Fe₂S₂ cluster and the c-type heme) lie in a plane parallel to that of the membrane, whereas the b hemes axis (cyclic path) is essentially perpendicular to it (9-12). As a consequence, only the cyclic path is electrogenic, as it involves the vectorial transfer of one charge across the membrane. This charge movement, in addition to PSI and PSII charge separation, is responsible for the generation of an electric field across the membrane () (19). The contributes with the pH (resulting from the activity of PSII and of cytochrome b₆f) to the generation of the electrochemical proton gradient (_H⁺) (19).

This model has been confirmed experimentally in the case of the cytochrome bc₁ complex, mainly on the basis of results obtained in the presence of the inhibitor antimycin (20), which is known to bind selectively to the Qi site (9-11, 21). However, these results cannot be transposed directly to the cytochrome b₆f complex for the following different reasons. (i) Antimycin is not effective in the case of the b₆f complex (21). Other inhibitors specifically affect the Qi site of the b₆f complex (e.g. 2-n-nonyl-4-hydroxyquinoline N-oxide; NQNO; 22), the mechanism of which seems to be similar to that of antimycin (23-24), although not identical (see a discussion in Ref. 17); (ii) the redox potentials of the b hemes are different in the bc₁ and the b₆f complexes (reviewed in Ref. 25). This difference probably explains why the equilibrium between the heme b_h and the quinone molecule is shifted in favor of the latter in the bc₁ complexes and of the former in the case of cytochrome b₆f complexes (16-17). This has a relevant consequence on the mechanism of quinone reduction at the Qi site; although the two electrons can be injected consecutively onto the quinone molecule in the case of the bc₁, the transfer must occur essentially in a concerted manner in the case of the cytochrome b₆f complex (16-17).

In this paper, we have studied the mechanism of cytochrome b₆ oxidation in intact cells of the green algae Chlamydomonas reinhardtii. We have observed an inhibition of the cytochrome b₆ oxidation rate (by a factor of ~10) when the membrane potential decay was accelerated by the addition of the ionophore FCCP, without substantial modifications of cytochrome f oxidation kinetics. The same phenomenon was observed also when the _H⁺ was reduced by changing the light intensity. Moreover, a partial recovery of this inhibition was obtained in the presence of ionophores when the potential was re-established by illumination with a train of actinic flashes fired at a frequency higher than its decay. Measurements of isotopic effects on the kinetics of cytochrome b₆ oxidation revealed a synergy between the effects of ionophores and the H₂O-D₂O exchange. We propose therefore, that protonation events influence the kinetics of cytochrome b₆ oxidation at the Qi site and that these reactions are strongly influenced by the light-dependent generation of a _H⁺.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains and Culture Media-- C. reinhardtii wild type cells derived from the strain 137C were kindly provided by the Laboratoire de Physiologie Membranaire et Moléculaire du Chloroplaste at the Institut de Biologie Physico-Chimique, Paris, France. They were grown at 27 °C in acetate-supplemented medium (26) under ~60 µE m² s¹ of continuous white light. They were harvested during exponential growth and resuspended at the required concentration in a 20 mM Hepes medium (pH 7.2) with 20% (w/v) Ficoll to avoid sedimentation. The ratio of the pellet and the resuspension buffer was usually 1:10. Alternatively, algae were resuspended in the same buffer containing D₂O (99.8% deuterium atom) and stirred for an hour. After a second centrifugation the pellet was resuspended in the same buffer with a pellet volume to resuspension buffer volume ratio of 1:20. No increase in the kinetic isotope effect was observed when the duration of the incubation in D₂O was increased. FCCP was purchased from Sigma.

Spectroscopic Measurements-- Spectroscopic measurements were performed at room temperature using a Joliot-type spectrophotometer as described in Joliot et al. (27). Actinic flashes were provided by a xenon lamp (3 µs at half-height) filtered through a Schott filter (RG 695). They were fired at a frequency of 0.15 Hz and were non-saturating (hitting ~25% of the centers) unless otherwise indicated. Algae were kept in the dark under argon atmosphere in a large reservoir connected to the measuring cuvette to ensure dark reduction of the plastoquinone pool.

The transmembrane potential was estimated by the amplitude of the electrochromic shift at 515 nm, which yields a linear response with respect to the electric component of the _H⁺ (28). Under the conditions employed here, the kinetics of the electrochromic signal displayed two rising phases previously described in Joliot and Delosme (29): a fast phase completed in less than 1 µs, associated with PSI and PSII charge separation (phase a), and a slow phase, which develops in the ms time range, associated with the turnover of the cytochrome b₆f complex (phase b). A third, slow decay was also present (phase c) that is mainly dependent on the passive ion leak through the membrane and the proton flux through the CF0-CF1 ATP synthase (29, 30). PSII absorption changes were prevented by a preillumination in the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) (10 µM) and hydroxylamine (1 mM) (31). Thus, the phase a was indicative of the sole PSI charge separation.

Cytochrome f redox changes were evaluated as the difference between absorption at 554 nm and a base line drawn between 545 and 573 nm. A small correction for the contribution of the electrochromic signal (5% of the signal observed at 515 nm) was made. Cytochrome b₆ redox changes were measured as the difference between the absorption at 564 nm and the same base line.

Protein Analysis-- Biochemical analysis was performed on thylakoid membranes obtained from cells that were incubated for 1 h at 30 °C and treated with 30 µM FCCP. Thylakoid membranes were purified after breaking the cells with a French press. The cytochrome b₆f complex was specifically solubilized from the thylakoid membranes with 30 mM Hecameg (2), and the solubilization supernatant was submitted to sucrose density gradient (10-30% sucrose, 20 mM Hecameg, 0.1 g/liter phosphatidylcholine, 20 mM Tricine-NaOH, pH 8.0, 3 mM MgCl₂, 3 mM KCl; see Ref. 2). SDS-polyacrylamide gel electrophoresis (12.5% acrylamide) followed by silver staining was performed on the fractionated gradient.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of Increasing Concentrations of FCCP on the Cytochrome b₆f Turnover Kinetics-- Fig. 1 shows the effects of increasing concentrations of the ionophore FCCP on the kinetics of the electrochromic signal (A), of cytochrome b₆ (B), and cytochrome f (C) redox changes. In Fig. 1A, the kinetics of the electrochromic signal is presented after normalization to the amplitude of the fast, PSI driven generation of the membrane potential (phase a). In these experiments indeed, the addition of hydroxylamine and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) blocked PSII activity and the consequent light generation of plastoquinol by PSII. Nevertheless, the plastoquinone pool was fully reduced in the dark by cellular metabolism (32), and the cytochrome b₆f Qo site was consequently saturated (see e.g. Ref. 33).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effect of increasing FCCP concentrations on the kinetics of the electrochromic signal (A), cytochrome b6 (B), and cytochrome f (C) redox changes. Algae were collected during exponential growth and resuspended in Hepes 20 mM, pH 7.2, with the addition of 20% (w/v) Ficoll to avoid sedimentation. They were illuminated with red flashes at the frequency of 0.15 Hz. 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) and hydroxylamine were added at the concentrations of 10 µM and 1 mM, respectively, to block PSII activity. FCCP concentrations were 1 µM (squares), 5 µM (circles), 15 µM (upward triangles), 30 µM (downward triangles), and 40 µM (diamonds). The chlorophyll concentration was ~ 50 µM. Traces in panels A and B represent a best fit using an equation described in Fig. 4.

The addition of the ionophore FCCP induced an acceleration of the membrane potential decay that was concentration-dependent. Thus, the decaying phase (phase c, see "Experimental Procedures") largely superimposed the rising one (phase b) in which amplitude was reduced accordingly. At higher concentrations of the ionophore, phase c became as fast as the phase b, and no net signal increase could be observed (Fig. 1A, diamonds).

In Figs. 1, B and C, the reduction phases of the cytochrome redox changes are shown as upward changes, whereas oxidation phases are shown as downward changes. The kinetics of cytochrome b₆ redox changes is presented in panel B. In the presence of 1 µM FCCP, a small signal increase (reflecting cytochrome b_l reduction) was observed followed by a large decrease signal. The latter is attributable to the oxidation of the b_h heme at the Qi site. Indeed, no redox signal changes are expected during the electron transfer between the b_l and b_h hemes, for their spectra are almost identical (Refs. 2 and 34; but see, however, Ref. 35). When the concentration of FCCP was increased from 1 µM to 40 µM, the kinetics of cytochrome b₆ oxidation became slower, whereas its reduction rate was substantially unaffected. As a consequence, the amplitude of the reduction phase increased at the expense of the oxidation signal. The inhibitory efficiency of FCCP was increased at higher temperatures, which decreased the incubation time required to observe such effects (not shown).

The kinetics of cytochrome f is reported in panel C. Both the oxidation and the reduction rates were not substantially affected by the addition of FCCP. We have verified that the signals measured in the presence of high concentrations of FCCP represented genuine changes of the cytochrome b₆ hemes; Fig. 2 shows the spectra of the redox changes measured in the 558-573-nm region in the presence of 1 µM (panel A) or 40 µM (panel B) of FCCP. In both cases, typical spectra of cytochromes b₆ were recorded (2, 34-35).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Light induced redox changes in the 558-573-nm spectral region measured upon single turnover flash illumination. Spectra were recorded 2 ms (squares) and 50 ms (circles) after the actinic flash illumination. FCCP concentration was 1 µM (A) or 40 µM (B). Other conditions are as in Fig. 1.

To test whether the inhibitory effect of FCCP was due to any artifact induced by the presence of large amounts of the chemical in the membranes (e.g. monomerization of the complex), we have examined some biochemical properties of cytochrome b₆f complexes extracted from thylakoid membranes treated or not with high concentrations of FCCP. The oligomerization state of the solubilized complex as well as its subunit composition were investigated by sucrose density gradient. The results are presented in Fig. 3, where it appears that FCCP did not alter either the oligomeric state cytochrome b₆f complex nor its composition. The treated complex migrated as a dimer (36), and all subunits comigrated, especially the Rieske protein. This is important evidence for the structural integrity of the complex, as it has been shown that in case of any perturbation of the complex, the Rieske protein is the first subunit to be lost (4, 33, 36).

View larger version (75K): [in this window] [in a new window]   Fig. 3.   Biochemical properties of cytochrome b6f complex upon treatment with FCCP. Thylakoid membranes were solubilized with Hecameg. The supernatant was then centrifuged on a 10-30% sucrose density gradient. Fractions were collected, analyzed by SDS-polyacrylamide gel electrophoresis, and silver-stained. Cytochrome f (Cyt f), cytochrome b6 (Cyt b6, poorly stained), the Rieske subunits (FeS), and subunit IV (SuIV) are indicated by arrows. b6f, purified b6f sample used as a marker. For other conditions, see "Experimental Procedures."

A more careful analysis of the data in Fig. 1 revealed a small but significant inhibition of both cytochromes b₆ and f reduction at very high FCCP concentrations (40 mM, Fig. 1, B and C, diamonds), suggesting the possible occurrence of nonspecific inhibition. To rule out the occurrence of any artifact in the observed phenomenon, we have repeated the same experiments using other ionophores (nigericin plus nonactin (37) and crown (34)). We found that they were equally effective in promoting the slowing down of cytochrome b₆ oxidation kinetics provided they were added at concentrations that substantially increased the membrane potential decay (not shown, see Fig. 6 below). On an other hand, the addition of the H⁺/K⁺ exchanger nigericin (37) at concentrations inducing the relaxation of the pH-only did not modify the kinetics of cytochrome b₆ oxidation at all (not shown).

Relationship between Transmembrane Potential and Cytochrome b₆ Oxidation Kinetics-- Taken together, these data suggest that the amplitude of the _H⁺ is able to affect the kinetics of cytochrome b₆ oxidation. This conclusion is reinforced by the data of Fig. 4, where the relationship between the rate of membrane potential decay and that of cytochrome b₆ oxidation is presented. Apparently there is a threshold effect; when the _H⁺ decay remained low as compared with the kinetics of cytochrome b₆ oxidation, the latter did not show any slowing, even when the rate of membrane potential decay was accelerated. However, when this rate became closer to that of cytochrome b₆ oxidation, an inverse proportionality could be observed. A new plateau level appeared for higher rates of the membrane potential decay.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Relationship between membrane potential decay and the rate of cytochrome b6 oxidation. Data were obtained by fitting data as in Fig. 1 using the following equation. (Eq. 1) This was done to improve the deconvolution of the electrochromic signal rise from its decay (see e.g. Ref. 38). Then rate constants were plotted. We checked that at low FCCP concentrations this procedure gave figures comparable with data previously obtained under similar conditions (38).

Two complementary experiments further confirmed the influence of the _H⁺ on the kinetics of cytochrome b₆ oxidation. In the first one we modulated the amplitude of the membrane potential by changing the intensity of the actinic light at a constant concentration of ionophores. This is shown in Fig. 5A, where the traces of the electrochromic signal measurements are shown. When the light intensity was decreased, the amplitude of the _H⁺ was diminished, because a smaller fraction of PSI reaction centers performed the charge separation. Under these conditions the cytochrome b₆ oxidation kinetics was slowed by a factor of 6-7, i.e. to an extent somewhat smaller but still comparable with that calculated in the presence of high concentration of ionophores (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of the transmembrane potential extent on cytochrome b6 oxidation kinetics. Effect of light intensity. Algae were illuminated with saturating (squares) or low intensity (~5%, circles) single turnover actinic flashes. Panel A: recordings of the electrochromic signals. Panel B, kinetics of cytochrome b6 redox changes. There kinetics have been normalized to allow a better comparison. Other conditions are as in Fig. 1.

In a second experiment, we increased the amplitude of the membrane potential in high ionophore concentration-treated samples by firing a series of flashes at a frequency that was sufficiently high to prevent its complete relaxation between two consecutive illuminations. According to the hypothesis mentioned above, an acceleration of cytochrome b₆ oxidation kinetic is expected upon rebuilding of the membrane potential. The results of this experiment are reported in Fig. 6; five flashes were fired to samples treated with low (squares) and high concentrations of ionophores (circles). The interval between two consecutive flashes (20 ms) was smaller than the rate of potential decay both in the low and the high ionophore concentration-treated samples (t_1/2 of ~ 500 ms and ~150 ms, respectively). Therefore, the _H⁺ was increased at each flash, as indicated by the increasing amplitude of the electrochromic signal after each flash (arrows in panel A). As expected, the effect of the flashes was larger in the low ionophore-treated samples than in the high ionophore-treated ones because of their different decay rates. In these experiments, a combination of nigericin plus nonactin was chosen as the high concentrated ionophore, since we found it easier to modulate the rate of potential decay using these compounds. Panel B reports the kinetics of cytochrome b₆ redox changes measured in the same conditions as in panel A. A marked difference in cytochrome b₆ oxidation rate could be observed between the low and high ionophore-treated samples after the first flash (panel C), whereas the difference largely diminished at the fifth flash, where the two rates became almost identical (panel D).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effect of the transmembrane potential amplitude on the kinetics of cytochrome b6 oxidation. Effect of illumination frequency. Algae were illuminated with a sequence of five saturating single turnover flashes (arrows), and the kinetics of the electrochromic signal (panel A) and of cytochrome b6 redox changes (panel B) were recorded. Panel C, kinetics of cytochrome b6 redox changes measured after the first saturating flash. Panel D, same kinetics as in C, measured after the fifth saturating flash. Traces have been superposed to allow a better comparison. Squares, 1 µM FCCP. Circles, 2 µM nigericin plus 4 µM nonactin.

The extent of the reduction and oxidation signals decreased from the first to the last flash in both the low and high ionophore concentration-treated samples (compare panels C and D). This is due to the fact that the interval between flashes was too short to allow a complete re-reduction of cytochrome b_h in the dark (34) (the b_l heme was always oxidized in our conditions because its dark reduction requires several minutes (34)). For this reason, the fraction of oxidized b₆f complexes (b_l⁺b_h⁺) increased during the flash series. These complexes are not expected to perform a net oxidation of cytochrome b₆ (see a discussion in Refs. 16, 32, and 38). Their increase, therefore, explains the reduction of the signal amplitude in both low and high ionophore concentration-treated samples. Obviously, the change in the relative proportion of oxidized and semireduced complexes is not expected to affect the overall rate of cytochrome b₆ oxidation (see e.g. Ref. 38). Consistently with this, these kinetics remained essentially the same in low ionophore concentration-treated samples (Fig. 6, panels C and D)

Isotopic Effect on Cytochrome b₆ Oxidation Kinetics-- It has been recently reported that the substitution of H₂O by D₂O in the reaction medium influences the kinetics of cytochrome b₆f (39). Although minor effects were observed on the rates of cytochrome f oxidation and of cytochrome f and b₆ reduction, a large slowing down of the initial step of the phase b was observed. This was interpreted as evidence for proton movement during the initial phase of the electrogenicity associated with cytochrome b₆f turnover (39). In this work, however, no data were presented concerning the oxidation kinetics of cytochrome b₆. We have therefore performed measurements of isotopic effects on this latter kinetics to understand whether proton diffusion-limiting steps were involved in this reaction.

The experiments have been performed essentially as in Deniau and Rappaport (39), and we have been able to reproduce the same results presented in the case of the phase b and cytochrome f redox changes (not shown) as well as the slowing down of cytochrome b₆ reduction (Fig. 7, A and B). In addition to this effect on the reduction, we observed a slowing down of the oxidation kinetics; the presence of D₂O in the reaction medium slowed the kinetics by a factor of ~2 (compare panels A and B). The isotopic effect increased when the algae were incubated with increasing concentrations of the ionophore FCCP; it was a factor of 3 when the concentration of FCCP was 15 µM and ~6 at 30 µM FCCP.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effect of increasing FCCP concentration on cytochrome b6 redox changes measured in the presence of H2O (A) or D2O (B). FCCP concentrations were 1 µM (squares), 15 µM (circles), 30 µM (triangles). Other conditions are as in Fig. 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of Membrane Potential on Cytochrome b₆ Kinetics-- We report here effects of increasing concentrations of the ionophore FCCP on the kinetics of cytochrome b₆f turnover. We found (Fig. 1) that high concentrations induce a selective slowing down of the kinetics of cytochrome b₆ oxidation (by a factor of ~10 upon increasing from 1 to 40 µM FCCP) without affecting the kinetics of cytochrome b₆ and of cytochrome f reduction (within a factor of 2). Consequently, the amplitude of the reduction phase of cytochrome b₆ kinetics was increased at the expense of the oxidation phase. In the 30-40 µM range, the observed kinetics did not show any net oxidation. High concentrations of FCCP have been previously used to dissipate the electrochemical proton gradient existing across thylakoid membranes in C. reinhardtii (40-41), and the obtained traces of cytochrome b₆ redox changes reported (42) closely resemble those measured here.

We believe that the decreased rate of cytochrome b₆ oxidation measured in the presence of FCCP is not due to a direct effect of the ionophore itself. Indeed, even if at very high concentrations, some small nonspecific effects can be evidenced (within a factor of 2, Fig. 1, B and C, diamonds), it was possible to reproduce its effects with other ionophores (see Fig. 6). The investigated biochemical properties of the complex remained unaffected upon incubation with 30 µM FCCP; the complex remained dimeric and contained all four high molecular weight subunits, including the Rieske protein, whose association with the complex is very sensitive to any structural perturbation (4, 33, 36). This hypothesis is in contrast with that proposed in previous studies (43-44) on the basis of measurements in isolated spinach thylakoids after treatments with the ionophore valinomycin (44). Klughammer et al. (44) indeed suggest that the slowing down of cytochrome b₆ oxidation results from specific binding of the ionophore to the complex itself, whereas this does not seem to be the case in our experimental conditions.

We can also rule out any influence of the redox state of the cofactors involved in the reaction in the observed phenomenon. Indeed, we have shown that changes in the redox state of the b_h heme, which can be modulated by changes in the illumination frequency, (34) do not modify the oxidation kinetics of the cytochrome b₆ (Fig. 6, see also Ref. 38). On the contrary, we propose that the slowed kinetics of cytochrome b₆ oxidation observed in the presence of high FCCP concentrations is due to the decrease of the _H⁺. This hypothesis is confirmed by the experiments performed in Figs. 4-6; there we show that the rate of cytochrome b₆ oxidation is not sensitive to the increase of membrane potential decay, provided that its amplitude remains unchanged during the turnover of cytochrome b₆f (Fig. 4). When the two rates become comparable, an effect is observed (Fig. 4). In addition, it is possible to mimic the effect of ionophores if the extent of the membrane potential is reduced by a decrease in the actinic light intensity (Fig. 5), and it is possible to recover a fast kinetic of cytochrome b₆ oxidation when the _H⁺ is reestablished by firing a series of flashes at a frequency higher than its decay rate (Fig. 6).

In principle, the two components of the membrane potential (the protonic one, pH, and the electric one, ) could be involved in the observed phenomenon. Different ionophores were able to reproduce the effect of FCCP provided that they increased the decay rate of the membrane potential, but the addition of the H⁺/K⁺ exchanger nigericin had no effect. It has been reported that this ionophore induces a partial conversion of the pH into (45). If the pH alone was responsible for the observed effects, one would have expected nigericin to be the most effective ionophore. Our results thus indicate that the presence of alone is sufficient to keep the kinetics of cytochrome b₆ oxidation in its native, fast state.

In green algae, it is known that a transmembrane potential can be established across the thylakoid membranes in the dark at the expenses of cellular metabolism (46). This potential could also influence the rate of cytochrome b₆ oxidation. However, we can rule out its involvement in our experiments, where FCCP was always present at concentrations (0.5-1 mM) sufficient to fully suppress it (45).

Mechanism of the Membrane Potential Effect on Plastoquinone Reduction by the Cytochrome b₆f Complex-- The oxidation of cytochrome b₆ is a complex reaction where both electron and proton transfer are involved (16-17). Three reaction steps concern electron transfer: cytochrome b_l reduction at the Qo site, interheme transfer between b_l and b_h, and the oxidation of the latter by a plastoquinone at the Qi site. A fourth one, the protonation of reduced plastoquinone, involves H⁺ transfer from the stroma to the Qi site.

Among them, only two (the interheme electron transfer and the protonation of reduced plastoquinone) occur in a plane parallel to the membrane potential, as inferred from the structure of the homologous cytochrome bc₁ complex (9-12). These steps are potentially influenced by the and represent possibly good candidates for the phenomenon reported here. However, we do not consider the interheme electron transfer as responsible for the observed enhancement of the rate of cytochrome b₆ oxidation (i.e. Fig. 6). Indeed (i) the target reaction step is sensitive to the H₂O-D₂O substitution (Fig. 7), i.e. it should involve protonation reactions (see e.g. Ref. 47). (ii) The presence of a should inhibit the interheme electron transfer by reducing its driving force.² (iii) In the absence as well as in the presence of , the rate of this reaction should be by far larger than that of cytochrome b₆ oxidation.² For very similar reasons, we can reject the involvement in the phenomenon described here of the Lavergne's factor "G" (Ref. 50; see also Ref. 32), a soluble cytochrome (32) that exchanges electrons with the b_h heme. Even if the equilibrium between the two is -sensitive, the electron transfer is accomplished in ~100 ms, i.e. at least 2 order of magnitude faster than the reactions observed here (Fig. 1).

In the past, the occurrence of a membrane potential-dependent back-reaction through the Qi site has been documented in the case of bc₁ complexes (51-54). The presence of this back-reaction has suggested that the interheme electron transfer is a reversible process. Thus it might be modulated by the also by affecting its equilibrium constant. Again, we consider this possibility rather unlikely for at least the two following reasons (i) The effect of on the equilibrium constant should be inhibitory rather than stimulatory, and (ii) the rate of the back-reaction is comparable with that of the forward process only under conditions that are not physiological (see e.g. Ref. 51), suggesting that the b_l to b_h interheme electron flow is probably a unidirectional process in native conditions.

Thus, we consider the possibility that the effects of reported here concern the electron transfer steps as very unlikely. Also an effect of on the rate constant of protonation is to reject for essentially the same reasons discussed above in the case of electron transfer. (i) The presence of should be inhibitory, and ii) this rate constant is also very high (see e.g. Ref. 55), at least when a continuous network of proton carriers is involved, as is the case in the quinone binding pockets (see a review in Ref. 56).

The proton transfer, therefore, is not the limiting step of cytochrome b₆ oxidation, unless the H⁺ concentration is not saturating. This is, however, likely to occur at the basic stromal pH (45). Therefore, a possible mechanism for the -mediated acceleration of cytochrome b₆ oxidation can be proposed based on the H⁺ availability at the Qi site. The , indeed, is expected to influence the pH at the membrane H₂O stromal interface, where protons would concentrate in its presence, to compensate the accumulation of negative charges (19). The increased H⁺ availability would enhance the rate of protonation at the Qi site in the presence of , as already observed in the case of other protein systems (57), and plastoquinone reduction would be limited by both electron and proton transfer steps at the Qi site. This would explain the rather low isotopic effect measured (within a factor of 2, Fig. 7, squares). Dissipation of the membrane potential would increase the local pH and consequently decrease the rate of plastoquinone protonation without directly affecting the electron transfer step. Protonation would then become the major limiting factor, explaining the increased isotopic effect up to a factor of 6 (Fig. 7, triangles). This value is usually considered as a strong indication that the rate-limiting step involves H-bond breaking or formation (47).

The data presented here clearly provide evidence for the effect of the membrane potential on the efficiency of cytochrome b₆ oxidation; independently of how the potential is decreased (by ionophores, Figs. 1 and 5, or by changing the actinic light intensity, Fig. 4), a parallel slowing down of the oxidation rate is observed.

However, a careful comparison of the results obtained with the two methods reveals stronger consequences of the membrane potential in high light-treated samples. This difference is not explainable by the hypothesis of a sole modulation by the membrane potential; neither can it be ascribed to specific effects of the high concentrations of ionophores employed under high light treatments (see above).

To us it suggests another possibility, i.e. that illumination might affect the turnover of the complex. Even if a direct influence of light is hardly explainable, consequences can be envisaged on the availability of the quinones. Indeed, evidences for a modification of the macroscopic structure of the thylakoids by illumination have already been reported (see e.g. Ref. 58). These changes might affect the diffusion of the quinone species (see a discussion in Lavergne and Joliot (59)), thus modulating the substrate availability at the Qo and Qi site. This effect is expected to have important consequences at the Qi site due to the scarcity of plastoquinone in the membrane under our anaerobic conditions.

Involvement of Cytochrome b₆f Qi Site in the Photosynthetic Control-- It is known that the _H⁺ couples electron transport to the generation of ATP (19). It also regulates the rate of the electron transfer into the cytochrome b₆f complex via the "photosynthetic control," a regulation mechanism analogous to the "respiratory control" observed in respiratory electron transport (reviewed in Ref. 60). The phenomenon is usually described as an inhibition of the rate of plastoquinol oxidation at the Qo site by the pH component of the _H⁺ (16-17, 19, 26, 45, 60). A mechanism for this phenomenon has been recently proposed in the case of the cytochrome bc₁ complex on the basis of its three-dimensional structure (61).

The results presented here, however, suggest a more complex scenario for the photosynthetic control, where the participation of the reactions occurring at the Qi site (i.e. the reduction of plastoquinone) should also be considered. In the presence of the _H⁺, indeed, the kinetics of the reactions that follow plastoquinol oxidation at the Qo site (cytochrome b₆ and f reduction) is slowed by the acid lumenal pH (see e.g. Ref. 45), whereas that of plastoquinone reduction at the Qi site (cytochrome b₆ oxidation) is accelerated by the concomitant generation of a (e.g. Figs. 1 and 5). Under these conditions, plastoquinol oxidation becomes rate-limiting. Upon dissipation of the _H⁺, either artificially by the addition of ionophores or via ATP synthesis, oxidation of plastoquinol at the Qo site is enhanced while its reduction at the Qi site is slowed down. The latter reaction becomes therefore the rate-limiting reaction in the whole turnover of the cytochrome b₆f complex (Fig. 1). This suggests an additional function of the _H⁺ in the modulation of the activity of this complex; it would balance the rates of the different catalytic reaction steps to optimize their concerted functioning.

	ACKNOWLEDGEMENTS

We thank Giorgio Forti for most valuable discussions and for his continuous support during the realization of this work. Fabrice Rappaport is also thanked for fruitful discussions and for kind help during measurements of isotopic effects on cytochrome b₆f kinetics.

	FOOTNOTES

^* This work was supported by the Consiglio Nazionale delle Ricerche.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of a doctoral fellowship from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica.

Recipient of a post-doctoral fellowship from the Max Planck Institute.

^** To whom correspondence should be addressed. Tel.: 39 02 26604423; Fax: 39 02 26604399; E-mail: giovanni.finazzi@unimi.it.

Published, JBC Papers in Press, June 23, 2000, DOI 10.1074/jbc.M002299200

² The driving force for electron transfer between the b_l and b_h hemes is expressed by G° = nFE°, where E° = 74 mV (2). When they are submitted to the light-induced electric field (), the G° is decreased according to G°= nF(E°), where  is the fraction of electrogenicity associated with the reaction.  represents the fraction of the membrane that is traversed by the electron transferred during the reaction. Thus, using a value of of 50-100 mV (48), a distance between the hemes of ~13 Å (edge to edge, Refs. 9-12) and a reorganization energy of  = 0.7-1 (49), it is possible to compute (49) the rate constant for the reaction in the 1.5-3 × 10⁶ s¹ in the absence of . This value decreased to 0.15-0.3 × 10⁶ s¹ in its presence. These values are to be compared with that calculated for cytochrome b₆ oxidation (0.1-0.3 × 10³ s¹, Fig. 1).

	ABBREVIATIONS

The abbreviations used are: PSI and PSII, photosystem I and II, respectively; E_m, redox midpoint potential; b_h, high potential heme of cytochrome b₆; b_l, low potential heme of cytochrome b₆; crown, dicyclohexyl-18-crown-6; FCCP, carbonylcyanide p-(trifluoromethoxy)phenylhydrazone; Hecameg, (6-O-(N-heptylcarvamoyl)-methyl--D-glucopyranoside; Qi, quinol-reducing site of cytochrome b₆f complex; Qo, quinol-oxidizing site of cytochrome b₆f complex; Tricine, N-tris (hydroxymethyl)methylglycine; I, reference transmittance; I, difference between the transmittance of the measuring sample and that of the reference.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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