quinol couples at the quinol oxidizing site of the cytochrome bf complex

BB

ELSEVIER

Biochimica et Biophysica Acta 1184 (1994) 251-262

etBiochl~Pic~a BiophysicaA~ta

Competition among plastoquinol and artificial quinone/quinol couples at the quinol oxidizing site of the cytochrome bf complex David M. Kramer ,,a, Anne Joliot b, Pierre Joliot b, Antony R. Crofts

a

a Biophysics Division, University of Illinois, Urbana, Illinois (USA), b Institut de Biologie Physico-Chimique, Service de Photosynth~se, 13, rue Pierre et Marie Curie, 75005 Paris, France (Received 2 July 1993; revised manuscript received 12 October 1993)

Abstract

We have investigated the interaction of plastoquinol (PQH2) , duroquinol (DQH 2) and duroquinone (DQ) with the PQH 2 oxidizing site (Qo site) of the chloroplast bf complex. In the absence of exogenous quinones or quinols, with an essentially fully reduced PQ pool, the half-time for reduction of cyt b upon a single-turnover actinic flash was approx. 2-2.5 ms, with an initial rate of about 250 s - 1. The same rate was found with an approx. 20% oxidized PQ pool, indicating that this rate is most probably at or near the Vmax for the reaction. When the PQ pool was reduced by addition of 100/zM DQH2, the rate of cyt b reduction was considerably slower, with a half-time of about 6 ms and an intial rate of about 100 s-1. Only at much higher concentrations of DQH 2 did the initial rate of cyt b reduction approach that found in samples where PQH 2 was the sole reductant. We concluded that, in the presence of 02, a fraction of the added DQH 2 was oxidized to DQ which acted as a competitive inhibitor of the Qo site. The slowed cyt b reduction kinetics were not observed when the production of DQ was prevented by excluding oxygen from the sample or by pre-reduction of the PQ pool by sodium dithionite. In strictly anaerobic samples titrated with a series of DQ and DQH 2 concentrations, where the PQH 2 pool was at all times essentially completely reduced, we were able to demonstrate a competitive interaction at the Qo site among PQH2, DQH 2 and DQ. By using an appropriate kinetic model consisting of an enzyme (the b f complex), two alternate substrates (PQH 2 and DQH 2) and one competitive inhibitor (DQ) - we were able to simulate the pre-steady state kinetics of cyt b reduction that resulted from this competition. From these simulations, we concluded that the Vma~ for oxidation of DQH 2 and PQH 2 were both about 250 s-1. The predicted binding constants for the species at the Qo site depended on the assumed values of the partition coefficients of DQ and DQH 2 into the thylakoid membrane. When estimated partition coefficients for the DQ and DQH 2 were introduced into the simulations, we were able to estimate that, the binding constant of PQH 2 to the Qo site was > 2.104 M -1. We concluded that, in native thylakoids, with a completely reduced PQ pool, essentially all Qo sites were occupied with PQH2, consistent with a relatively tight binding. Approx. 3/xM of added DQ is expected to displace PQH 2 from half of the Qo sites. DQH 2 can displace nearly all of the DQ from the sites, but only at high added concentrations. At the high concentrations of DQH 2 typically employed in electron transfer assays typically 0.5-1 mM - nearly all turnovers of the complex occur at the expense of DQH 2. At lower concentrations, in the presence of 02, competitive inhibition by DQ can severely affect experimental results. We have also found that decyl-ubiquinol (dUQH 2) is a substrate for the b f complex, but with the product of partition coefficient into the membrane and binding constant into the Qo site greater than that of DQH 2 and DQ.

Key words: Chloroplast; Cytochrome b f complex; Electron transport; Plastoquinone; Semiquinone; Duroquinone; Duroquinol

* Corresponding author (present address at (b): Paris). Fax: +33 1 40468331; e-mail: [email protected]. Abbreviations: Ar, Argon gas; bf, the cytochrome bf complex, or the plastoquinol-plastocyanin oxidoreductase; cyt, cytochr0me; cyt bh, the high potential b cytochrome of the cytochrome bf complex; cyt bl, the low potential b cytochrome of the cytochrome bf complex; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; dUQ, decyl-ubiquinone; dUQH2, decyl-ubihydroquinone; DQ, tetramethyl-p-benzoquinol or duroquinone; DQH2, tetramethyl-p-benzoquinol or duroquinol; DMSO, dimethylsulfoxide; Eh, the ambient redox potential, Era.x, the midpoint potential, the midpoint potential at a particular pH, x; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesuifonic acid; FeS, the Rieske iron sulfur center of the cytochrome bf complex; MV, methyl viologen; NQNO, 2-n-nonyl-4-hydroxyquinolineN-oxide; PC, plastocyanin; PS II, Photosystem II; PS I, Photosystem I; PQH2, plastoquinol; PQ, plastoquinone; PQ-, plastosemiquinone; QB, the secondary plastoquinone binding site of photosystem II; Qo, the plastoquinol oxidizing site of the cytochrome bf complex; Qi, the plastoquinone reducing site of the cytochrome bf complex; TMQ, trimethyl-p-benzoquinone; TMQH2, trimethyl-p-benzohydroquinone. 0005-2728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0005 -2728(93)E0186-T

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1. Introduction

The function of electron transport chains is typically investigated by observing the turnover of the partial reactions catalyzed by the various enzyme complexes of the chain. Often, artificial donors, acceptors and redox mediators are employed to isolate a particular set of partial reactions and to establish well determined and repeatable experimental conditions for study. Usually, for kinetics studies, the so-called 'poise and pulse method' is used, where a redox mediator allows the redox state of the experimental system to equilibrate with the environment and measuring electrodes on a time scale convenient for experimentation, but not so rapidly that the mediation interferes with the turnover of the complex in the time scale of the observation (for review, see Ref. [1]). In steady-state experiments, artificial donors and acceptors are usually added to establish conditions where the turnover of the enzyme can be studied without concern for depletion of substrate or accumulation of product. These compounds should be competent alternate substrates for the complex and should have no interfering side reactions or effects. Deviations from these criteria that are not accounted for, will result in artifacts. It is difficult, in most cases, to establish these criteria, especially if the turnover of the complex solely in the presence of the native substrate alone is not well characterized. Many studies of the electron transfer chain of higher plant chloroplasts, particularly studies of the cytochrome bf complex, have used tetramethyl-p-benzoquinol (Duroquinol, DQH 2) either as electron donor to the plastoquinone pool, as an oxidizable substrate, or, together with its oxidized form, duroquinone (DQ), as a redox mediator (for examples, see Refs. [2-5]). However, some important questions remain about the function of the electron transport chain of chloroplasts in the presence of DQH 2 and DQ, in particular whether DQH 2 is a substrate and whether either DQH 2 or DQ modifies the behavior of the chain seen when only the natural substrate plastohydroquinone (plastoquinol, PQH 2) is present as electron donor. Early studies [2,3] concluded that DQH 2 donated electrons to the PQ pool, which in turn acted as donor for turnover of the bf complex. However, it appears that DQH 2 reduction of the PQ pool is too slow to account for the steady-state turnover supported by the donor [5,6]. In a recent study by Rich et al. [5], the reduction of the PQ pool by DQH 2 was determined by measuring the area above the fluorescence induction curve. They concluded that DQH 2 reduced the PQ pool only slowly (a half-time of about 10 s upon addition of 1 mM DQH2), due to a lack of measurable quinol-quinone transhydrogenase activity. In similar experiments (Kramer, D.M., Bechman, G. and Crofts, A.R., unpublished), after taking into account complica-

tions due to oxidation of PQH 2 and DQH 2 in the presence of 0 2 and the quenching of chlorophyll fluorescence by DQ, it was concluded that the rate of reduction of the PQ pool by added DQH2 was more rapid than that suggested by the work of Rich et al. [5]. However, the most rapid observed rate (somewhat less than 5 s half-time for reduction of the PQ pool by addition of 100 /xM DQH 2) was still insufficient to explain the rapid steady-state turnover of the bf complex (see Discussion) with similar concentrations of DQH 2. Therefore, it is concluded that DQH 2 is most likely a substrate for turnover of the bf complex. In this work, we extend our studies of the interaction of DQH 2 with the electron transfer chain, using pre-steady state kinetics to probe the interaction of DQH2, DQ and PQH 2 with the bf complex. In order to explain our data, it was necessary to include DQ as species interacting at the Qo site. In addition to explaining the paradoxical results from earlier experiments, these investigations allowed us to estimate the relative binding constants of PQH2, DQH 2 and DQ for the quinol oxidizing site of the bf complex.

2. Materials and methods

2.1. Preparation of thylakoids Thylakoids were prepared by a modification of the procedure used by Moss and Bendall [6]. Approx. 500 g of market spinach was washed and de-veined, then ground in a blender in medium containing 343 mM sorbitol, 0.4 mM KC1, 0.04 mM EDTA and 50 mM Hepes buffer (pH 7.6), at 4°C, and filtered through eight layers of Miracloth (Calbiochem). The filtrate was centrifuged at 2000 × g for 90 s at 4-8°C. The supernatant was discarded and the pellet was re-suspended to a chlorophyll concentration of about 4 m g / ml in a buffer containing 330 mM sorbitol, 10 mM KC1, 1 mM EDTA, 5 mM MgC12 and 50 mM Hepes (pH 7.6). After determination of chlorophyll concentration [7], 10% DMSO was added as a cryoprotectant and aliquots containing 1 mg chlorophyll were frozen at -70°C for up to 2 months. After freezing, the chloroplast envelope was ruptured since essentially all thylakoids were rapidly accessible to ferricyanide (data not shown). No differences in measured kinetics were noted when a short osmotic shock, followed by centrifugation and re-suspension in medium, was given prior to freezing. Samples frozen with 10% DMSO did not show any loss of activity for up to 3 months at -70°C. For use in experiments, samples were thawed at 0-4°C, and suspended in the re-suspension buffer (ph 7.6) with 10% Ficoll and 1/~M gramicidin as an uncoupler.

D.M. Krameret aL/ Biochimica et BiophysicaActa 1184 (1994)251-262 For studies on the interaction of the PQ pool with the Qo site of the bf complex we chose a procedure, similar to that first introduced by Velthuys [8,9], to reduce the PQ pool. Thylakoids were suspended to a photo-oxidizable cyt f concentration of 41 nM (approx. 25-30 /xg chlorophyll per ml sample) in suspension medium previously bubbled with Ar for a minimum of 5 min to exclude oxygen. Measurements of photooxidizable cyt f concentrations were made by following the maximal oxidation of cyt f after a train of 15 actinic flashes in suspension medium, at pH 7.6, in the presence of 10 /xM DCMU, 1 mM sodium ascorbate and 10 # M methyl viologen, and quantified using the deconvolution procedure of Joliot and Joliot [10] and the extinction coefficients of Rich et al. [11]. Purged samples were illuminated for 20 s with approx. 200/zE m - 2 S-1 white light from an incandescent light source. In the absence of acceptor or DCMU, the illumination was sufficient to reduce the PQ pool and the high potential chain components of the intermediate electron transfer chain (i.e., plastocyanin and the iron-sulfur center and cytochrome f of the cytochrome bf complex). Samples were then placed in complete darkness and 10 /zM DCMU (to block further turn-over of PS II), 5/zM NQNO (to slow the re-oxidation of cyt b, see Ref. [4]) and 10 /zM MV (as an electron acceptor for PS I) were added in the dark with thorough mixing. The samples were then introduced into the sample holder of the double-flash kinetic spectrophotometer and equilibrated in the dark for 20 s. Additional experiments (Kramer, D.M., Joliot, A., Joliot, P. and Crofts, A.R., unpublished data) showed that, after this procedure, more than 95% of the PQ pool was reduced. All experiments were performed at 23°C.

2.2. Spectrophotometric measurements The double-flash kinetic spectrophotometer was constructed in-house, based on the design suggested by Joliot et al. [12] and Joliot and Joliot [13] with modifications as described in Kramer [14]. Redox potentiometric measurements were made with a glassy carbon vs. standard calomel electrode, calibrated with saturated quinhydrone (Kodak) at pH 7.0. We were unable to obtain reproducible measurements of the poise of the D Q : D Q H 2 couple with a platinum sheet electrode, presumably because DQ and DQH 2 react poorly with the Pt surface. By measuring the E n of anaerobic samples of suspension buffer with various ratios of DQ to DQH 2, we estimated the Em,7(DQ/DQH 2) at approx. + 65 mV, with an n = 2 redox chemistry and a pH dependence of approx. - 6 0 mV per pH unit, comparable to literature values between 57 and 70 mV [15,16].

253

2.3. Deconvolution of kinetic traces Kinetic traces of flash-induced absorbance changes were deconvoluted by the method of Joliot and Joliot [10]. Concentrations of P700 and the bf complex were determined by measuring the extent of photo-oxidizable P700 and cyt f, respectively after a train of 15 actinic flashes (at 50 Hz), in samples treated with 10 /zM DCMU, 10/xM methyl viologen and 1 mM sodium ascorbate. Extinction coefficients for these species were as described by Rich et al. [11]. 2.4. Materials Duroquinone and decyl-ubiquinone were purchased from Sigma and reduced to the quinol forms by the method of Izawa et al. [3]. Key experiments were also performed using duroquinol reduced and purified as in [5], a gift from Dr. G. Bechman, with similar results. NQNO was a gift from Professor J. Whitmarsh. 2.5. Computer-aided fitting of rate equation parameters to measured kinetics A computer program was written using QuickBasic (Microsoft) to fit parameters of the rate equation derived in the Discussion to measured kinetic parameters. The fitting algorithm allowed any combination of parameters in the rate equation to be used as free fitting parameters or to be set as constants. An iteration of the algorithm consisted of setting arbitrary initial values, after which one of the free fitting parameters was varied by 1% at a time in the direction that decreased the sum of squares of the differences between the data points and the predicted values from the simulation. When no improvement could be gained, the next free fitting parameter was varied. When all fitting parameters were varied, the iteration was repeated, until no improvement was gained. Local minima were tested by using various initial values and by changing the order of parameter fitting for each iteration of the program.

3. Results

3.1. Kinetics of reduction of cyt b As an indicator of the turnover of the complex, we measured the rate of reduction of cyt b in the presence of NQNO. The rate of cyt f re-reduction is complicated by overlapping oxidation and reduction phases and by rapid equilibration with other redox components in the high potential chain (see [14,17]) and is therefore not a good indicator of pre-steady state turnover rate. We concluded that the rate of cyt b

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reduction measured 2 ms or earlier after the actinic flash was a good approximation of the initial rate by the following reasoning. We estimated the ratio of PS I:cyt f in our preparation to be between 1.0:1.0 to 1.2:1.0 (see above). Therefore, we expect that, after one saturating single-turnover actinic flash to PS I, approximately one positive charge per bf complex would have been introduced into the high potential chain. We then expect an average of about one electron per bf complex to be delivered to the low potential chain after this flash. When such a flash is given to thylakoids with a fully reduced PQ pool in the presence of NQNO a substantial fraction of reduced cyt b (0.6-0.7 heme per bf complex) is accumulated (see Fig. 1, trace A). We would not expect to accumulate a large amount of reduced cyt b if the rate of its re-oxidation were rapid. The data points taken in this time range (see inset in Fig. 2) showed a reasonably linear rate, indicating that product inhibition (e.g. accumulation of reduced cyt b and high potential chain components) most probably did not significantly slow the forward rate of the reaction during this time (for further discus-

0

10

20

30

40

50

400

800

1200 1600

Time (ms) Fig. 1. Effects of reduction of the plastoquinone pool by various methods on the kinetics of flash-induced reduction and re-oxidation of cyt b. Spinach thylakoids were suspended at a concentration of 40 nM cyt f (see Materials and methods) in medium containing 330 mM sorbitol, 10 mM EDTA, 5 mM MgCI 2 and 50 mM Hepes (pH 7.6) in the presence of 5/zM NQNO, 10/~M DCMU and 5/zM MV at 23°C. The samples were given a saturating, single-turnover actinic flash at time zero and the kinetics of cyt b reduction and re-oxidation were monitored by absorbance spectroscopy as described in the Materials and methods section. To achieve more complete kinetic information in the time range from 1-8 ms, the traces were constructed from 2-3 composite traces with a range of time intervals between the actinic flash and the first measuring flash. The PQ pool was fully reduced using the following tec.hniques: trace A, by pre-illumination under anaerobic conditions, without DCMU or PS I electron acceptor, followed by addition of these in the dark, as described in the text; trace B, by aerobic addition of 100 ~M DQH2; trace C, by titration, under anaerobic conditions, with sodium dithionite to the point where cyt b reduction was most rapid, but before measurable reduction of cyt b in the dark, as described in the text; trace D, as in trace C, but with the anaerobic addition of 100 /.~M DQH 2 (trace D). The inset shows an expanded time base for the above traces.

1.5,

0.

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10

20

30

40

~ 50

~ 400

~ 800

1 2 0 0 1600

Time (ms) Fig. 2. Flash-induced redox reactions of cyt b as a function of DQH 2 concentration, in the presence of DQ. Anaerobic spinach thylakoid suspensions (as in Fig. 1, but in medium bubbled with Ar for 5 min prior to the experiment and kept under flowing Ar) in the presence of 5 /zM NQNO, 10 /xM DCMU, 5 /zM MV and 8 izM DQ and various concentrations of DQH2, were given a saturating, singleturnover actinic flash at time zero and the kinetics of cyt b reduction and re-oxidation were monitored by absorption spectroscopy as described in the Materials and methods section. The amount of added DQH 2 for the traces was as follows: (A), 2.5/~M; (B), 10 IzM; (C), 42.5 ~M; (D), 85 ~M; (E), 170/xM; (F), 340 ~M.

sion, see Discussion). Furthermore, we have measured the kinetics of cyt f oxidation in our preparation to determine if this rate could significantly affect the measurements of initial rates of cyt b reduction (data not shown). Under conditions where cyt f re-reduction was prevented, either by addition of DCMU in the presence of 1-5 mM sodium ascorbate, or upon addition of 10/xM stigmatellin in the presence of 100/xM duroquinol, we found half-times for cyt f oxidation of between 125 and 150/~s, with a short lag in the onset of about 20-35 /xs, similar to values previously found (e.g., see Refs. [17,18]). Thus, the rate of cyt f oxidation was unlikely to have caused significant errors in the analysis of the initial rate of cyt b reduction.

3.2. Flash-induced reduction of cyt b with a reduced PQ pool, in the presence and absence of added DQH 2 Fig. 1 shows flash-induced cyt b changes in thylakoid suspensions after the PQ pool was reduced by various techniques. In all traces, 5 I~M NQNO was added to slow the re-oxidation of cyt b, 10 I.~M MV was added as a P S I acceptor and 10 I-~M DCMU was added to prevent further reduction of the PQ pool by PS II. When DQH 2 was used, 3 min dark time was allowed before each actinic flash to allow complete equilibration of the donor with the PQ pool. In trace A, the PQ pool was reduced by pre-illumination as described in the Materials and Methods section. When this treatment was performed in suspensions well purged of oxygen, leading to more than 95%

D.M. Kramer et al. / Biochimica et Biophysica A cta 1184 (1994) 251-262

reduction of the PQ pool (see above), we measured the maximal rate of cyt b reduction, with a half-time of 2-2.5 ms and an initial rate - measured by a best-fit straight line through the origin and the 1 and 2 ms points, approximating the initial slope of about 250 s-1. When a second flash was given to this suspension 10 s after the first, the cyt b kinetics were nearly identical to those after the first flash (data not shown). Since approx. 1 PQH 2 molecule was oxidized per bf complex and the total PQ pool is estimated to be approx. 5-6 P Q / b f complex (see Refs. [19-24]), we expect the second actinic flash to have probed the reduction of cyt b in the presence of an approx. 16-20% oxidized PQ pool. The lack of significant effect of this change in the poise of the PQ pool, suggests that the Qo sites were saturated by PQH 2 over this range of PQH 2 concentrations (see Discussion). In other experiments (Kramer, D.M., Bechman, G. and Crofts, A.R., unpublished data) it was found that, under anaerobic conditions, even low concentrations (e.g. lower than /xM) of added DQH 2 fully reduced the PQ pool (see Discussion). Under the conditions used in trace B of Fig. 1 (aerobic sample, pH 7.6), we concluded that addition of 100 /~M DQH 2 to the suspension fully reduced the PQ pool within the dark adaptation period of 3 min, since no effect on the area above fluorescence induction curves was observed upon addition of 10/.~M DCMU (data not shown). In contrast to the results with pre-illuminated thylakoids (where PQH 2 was the sole donor to the complex, see Fig. 1, trace A), when the PQ pool was reduced by addition of 100/zM DQH 2, a much slower rate of cyt b reduction was observed upon flash excitation (see Fig. 1, trace C). The half-time increased to 6 ms and the initial rate slowed to about 100 s -1. The rate of re-oxidation of cyt b also appeared to be slower in thylakoids reduced by DQH 2, though the exact degree of slowing is difficult to measure in these traces due to overlapping reduction and re-oxidation phases. The initial rate of cyt b reduction, as well as its re-oxidation, increased with increasing concentrations of added DQH 2, even at concentrations of DQH 2 in excess of 1 mM (data not shown). Since the PQ pool was essentially fully reduced upon addition of 100 tzM DQH2, we concluded that the anomalously slow reduction kinetics of cyt b reduction in the presence of DQH 2 were most probably due either to the presence of the DQH 2 itself, or to the presence of a small concentration of DQ that would have inevitably been formed upon reduction of oxidants in the suspension (the PQ pool, other quinones, O 2 and etc.). To distinguish between these possibilities, we devised a procedure to pre-reduce all oxidants in the suspension with midpoint potentials approximately equal to or lower than that of the PQ pool. Thylakoid

255

suspensions containing 10/.LM MV, 5 IzM NQNO and 10 /~M DCMU were de-gassed with Ar for at least 5 min and small aliquots of a 50 mM Sodium dithionite solution (dissolved in Ar-purged H20) were added. This experiment required redox conditions where the PQ pool would be fully reduced but cyt b chain would be fully oxidized. Therefore, 2 min after each addition of dithionite, the redox state of cyt b in the dark was monitored by scanning the absorbance spectra between 545-575 nm. The redox state of the PQ pool was monitored by measuring kinetics and extent of flash-induced cyt b reduction kinetics. After addition of 25 /xM dithionite, cyt b-559 and cyt f were fully reduced, but the PQ pool was only fractionally reduced as indicated by the kinetics of cyt b and f reduction, which were similar to those observed in pre-illuminated samples, but after a train of several actinic flashes to partly oxidize the PQ pool (data not shown). Between 75-100 /xM dithionite was required to fully reduce the PQ pool. Above 120 tzM, significant reduction of cyt b was observed (data not shown). After addition of 100 ~M dithionite, the initial rate of cyt b reduction was nearly identical to that measured in pre-illuminated thylakoids with a nearly fully reduced PQ pool (Fig. 1, trace C). No effects on cyt b reduction kinetics were observed when 100/~M DQH 2 was added to samples with a PQ pool fully reduced by titration with dithionite. Thus, we concluded that the presence of DQ and not that of DQH 2, resulted in the slowed cyt b reduction kinetics. 3.3. Reduction o f cyt b in the presence o f Duroquinone and Duroquinol

To further characterize the interaction of DQH 2, PQH 2 and DQ with the Qo site of the complex, we measured the initial rate of cyt b reduction following an actinic flash in the presence of controlled amounts of DQH 2 and DQ. It was necessary to minimize the amount of DQ produced upon addition of DQH 2. To do this, we purged the medium of 0 2 by bubbling with Ar gas for at least 5 min. After this treatment, cyt f, PC and cyt b559 were observed to have become reduced, most probably by an endogenous reductant (data not shown). The PQ pool was not reduced under these conditions since, in the presence of 10 /.~M DCMU and 5 /xM NQNO, flash actinic illumination did not lead to reduction of cyt b or appreciable re-reduction of cyt f (data not shown). After this treatment, a small, controlled amount of DQ was produced upon addition of DQH2, as judged by relatively rapid flash-induced cyt b reduction kinetics (see Fig. 3). Similar results could be obtained by addition of 50 /zM sodium ascorbate to samples dark-adapted under flowing (but not bubbling) Ar (data not shown). I n order to estimate the small concentration of DQ pro-

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D.M. Kramer et al. / Biochimica et Biophysica Acta 1184 (1994) 251-262

A\

ra

4 O.

0.6

~

0.4

/

/

/ I

/ /

7

6G

o 035

~ / - t

030

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1

\

/ 1o-7

0.(3 0

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I

50

100

I

I

150 200 [DQH2] ( I-tM')

lo ~,

lO-5

r,~p

lo ~'

1o.3

I

I

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250

300

350

Fig. 3. Dependence of initial rate of cyt b reduction on the competition among PQH 2, DQH 2 and DQ for the quinol oxidizing (Qo) site. The initial rates of cyt b reduction after a saturating single-turnover actinic flash were determined from experiments similar to those presented in Fig. 2. The slope of a straight line drawn through the first three time points (1-2 ms) of the cyt b kinetic traces was used as a measure of the initial rate. With the exception of trace A (in which the PQ pool was prereduced by dithionite titration, as in Fig. 1, trace C), a small amount of DQ (less than 0.5 /zM in well deoxygenated samples) was produced after addition of DQH 2, upon reduction of the PQ pool, so the indicated final concentrations of DQ and DQH 2 were estimated by comparing the measured ambient redox potential with that expected without DQ production (see text). The solid curves represent the best global fit to the kinetic model presented in the text. The partition coefficients for DQ and DQH 2, the concentration of PQ (PQH 2) and the volume of the thylakoid membrane were assumed from literature values (see text). The free variables for the global fit were the disassociation constants for binding of PQH 2, DQH 2 and DQ to the Qo site. Best fits were obtained when Vm~ for oxidation of DQH 2 was approximately equal to that for oxidation of PQH 2. The inset shows the dependence of the root-mean-square (rms) error of the best global fit to the data on the fixed value of the disassociation constant for binding of PQH 2 to the Qo site. Trace A, no DQ added or produced (see Fig. I, trace C for details); trace B, 1.4/~M DQ; trace C, 2.9/zM DQ; trace D 5.4 /zM DQ; trace E, 7.9/~M DQ; trace F, 16.4/zM DQ; trace G, 27.4 /zM DQ.

duced by reduction of the PQ pool, we measured the E h of the suspension and calculated the concentrations of D Q and D Q H 2 based on this measurement and the midpoint potential of the D Q : D Q H 2 couple (see Materials and methods) at the suspension pH. Under the conditions of the experiments in Fig. 2, only a small concentration (slightly less than 0.5 ~M, or approximately the concentration of P Q in the cuvette) of D Q was produced by oxidation of D Q H 2 upon its addition to the suspension. As long as the samples were maintained under strictly anaerobic conditions and were only exposed to a limited number of actinic flashes (less than 25 total), the amounts of D Q produced during the experiments were small and could readily be estimated. For data analysis, the total concentrations of D Q and D Q H 2 were corrected for the amount of D Q produced upon addition.

When the initial rate of cyt b reduction was plotted against the concentration of added D Q H 2, at different initial D Q concentrations, a family of curves was obtained (Fig. 3, traces B - H ) . The initial rate increased with increasing concentrations of D Q H 2 at all non-zero concentrations of DQ, but the slope of the increase was lower with higher concentrations of DQ. This family of curves is interpreted as a reflection of the competition for the active site between PQH2, D Q H 2 and DQ. In all cases, the rate at high [DQH 2] appears to approach the maximal rate, obtained with 'pre-illuminated' thylakoids, or with thylakoids with a pre-reduced PQ pool (i.e., with P Q H 2 as sole reductant) (see Fig. 3, trace A). This most likely indicates that Vmax for turnover of the complex with D Q H 2 as substrate is roughly equal to that with the native P Q H 2. By extrapolating to zero concentration of DQH2, we can estimate the rate of turnover of the complex in the presence of various concentrations of DQ, with P Q H 2 as the sole reductant. We conclude that at approx. 3 /xM D Q displaced P Q H 2 from about half of the Qo sites. Therefore, P Q H 2 as well as D Q H 2 competed for the Qo site. A more refined analysis, achieved by fitting the data to a kinetic model (see Discussion) gives a similar estimate.

4. Discussion 4.1. Reduction o f cyt b by P Q H 2 or D Q H 2 and the competitive inhibition o f this reaction by D Q

As discussed above, under proper conditions, addition of low concentrations of D Q H 2 completely reduced the PQ pool. It was surprising, therefore, to find that, under these conditions, the half-time and initial rates for cyt b reduction after flash excitation were considerably slower in the presence of D Q H 2 than those observed in the presence of a fully reduced PQ pool without additions (i.e., in 'pre-illuminated' thylakoids, see Fig. 1 traces A and B). It is reasonable to suggest that the slowing of the initial rate of cyt b reduction after a flash was the result of the addition of a substance which competes with P Q H 2 for the Qo site. Two possible competitors were added: D Q H 2 and the low concentrations of D Q formed after reduction of the PQ pool by D Q H 2. Since maximal initial rates of cyt b reduction were observed under anaerobic conditions and when the PQ pool was pre-reduced (see Figs. 1 and 3), in the presence or absence of D Q H 2, where production of DQ was prevented, we conclude that DQ, rather than DQH2, is a competitive inhibitor of the Qo site. Also consistent with this interpretation, is the finding that the initial rate of cyt b reduction decreased with increasing concentrations of D Q (see Figs. 2 and 3) and, in the presence of DQ, the initial

D.M. Krameret al. / Biochimica et BiophysicaActa 1184(1994)251-262 r a t e o f cyt b r e d u c t i o n i n c r e a s e d w i t h i n c r e a s i n g conc e n t r a t i o n s o f D Q H 2 (see Fig. 3). It is r e a s o n a b l e to c o n c l u d e t h a t D Q H 2 is a s u b s t r a t e for t h e bf c o m p l e x and, at high c o n c e n t r a t i o n , c a n d i s p l a c e D Q f r o m t h e catalytic site.

4.2. Implications for the binding of PQH 2 to the Qo site W h e n t h e P Q p o o l is r e d u c e d e i t h e r by pre-ill u m i n a t i o n to PS II, o r by t i t r a t i o n with d i t h i o n i t e (see Fig. 1), t h e initial r a t e o f cyt b r e d u c t i o n was m e a s u r e d at a b o u t 250 s - t . Since t h e r a t e o f cyt b r e d u c t i o n at high c o n c e n t r a t i o n s o f D Q H 2 a p p r o a c h e s t h e s a m e m a x i m a l o b s e r v e d r a t e , w e e s t i m a t e t h a t t h e catalytic r a t e o f o x i d a t i o n o f b o u n d D Q H 2 is similar to t h a t o f b o u n d P Q H 2. This is s u p p o r t e d by t h e fact t h a t a d d i tion o f D Q H 2 to t h y l a k o i d s with a p r e - r e d u c e d P Q p o o l h a d little effect on t h e cyt b r e d u c t i o n kinetics. Since w e w o u l d e x p e c t an i n c r e a s e in initial r a t e o f cyt b r e d u c t i o n if v a c a n t Qo sites w e r e p r e s e n t b e f o r e a d d i t i o n o f D Q H 2, as t h e y b e c a m e o c c u p i e d with t h e a l t e r n a t e s u b s t r a t e , D Q H 2, t h e m o s t r e a s o n a b l e conclusion is t h a t with a fully r e d u c e d P Q p o o l a n d no f u r t h e r a d d i t i o n s , e s s e n t i a l l y all Qo sites w e r e o c c u p i e d with P Q H 2. This i m p l i e s a relatively tight b i n d i n g o f P Q H 2 at t h e Qo site.

257

4.3. Simulation of the competition among DQ, DQH 2 and PQH2: implications for the mechanism of the bf complex U s i n g t h e following a s s u m p t i o n s , a s i m p l e m o d e l c a n b e c o n s t r u c t e d t h a t a d e q u a t e l y explains t h e d a t a in Fig. 3. Since t h e c o n c e n t r a t i o n o f P Q H 2 was c o n s t a n t for all d a t a p o i n t s in t h e curves in Fig. 3 (i.e., t h e a m b i e n t p o t e n t i a l was always sufficient to essentially fully r e d u c e the P Q pool, see above), we can a s s u m e t h a t all c h a n g e s in t h e r a t e o f cyt b r e d u c t i o n w e r e t h e result of c h a n g e s in t h e c o n c e n t r a t i o n s o f D Q H 2 a n d D Q . F u r t h e r m o r e , t h e s a m e c o n c e n t r a t i o n of oxidizing e q u i v a l e n t s w e r e i n t r o d u c e d into t h e high p o t e n t i a l c h a i n for e a c h e x p e r i m e n t (i.e., a b o u t 1 positive c h a r g e p e r P S I ) . T h e r e f o r e , as long as t h e initial r a t e o f cyt b r e d u c t i o n is m e a s u r e d b e f o r e significant c h a n g e s in t h e c o n c e n t r a t i o n o f o x i d i z e d high p o t e n t i a l c h a i n c o m p o n e n t s have o c c u r r e d , we c a n simplify t h e r e a c t i o n to o n e o c c u r r i n g at a single active site (i.e., t h e o x i d a t i o n o f a quinol at t h e Qo site) with two p o s s i b l e s u b s t r a t e s for t h e site - P Q H 2 a n d D Q H 2 - a n d o n e c o m p e t i t i v e i n h i b i t o r ( D Q ) . T h e s i m p l i f i e d kinetic m o d e l is illust r a t e d in S c h e m e 1. T o s i m u l a t e t h e kinetics, w e a s s u m e d t h a t t h e r a t e limiting s t e p in t h e o x i d a t i o n o f e i t h e r P Q H 2 o r D Q H 2 u p o n flash e x c i t a t i o n was t h e o x i d a t i o n o f b o u n d sub-

E

PQH2 :

Km,p

= E'PQH2

DQ~ PQH2 ~

[

~

kp

bh Km,D

E'DQ

bI DQ ~

~.?

FeS

~P

E'DQH2

f

DQ

kD

DQH2

Scheme 1. Model for competition of parallel substrates (PQH 2 and DQH2 ) and an inhibitor (DQ) for the Qo site of the cytochrome bf complex. The model is used to explain the cyt b reduction kinetics in the presence of PQH2, DQH 2 and DQ. The initial rate of cyt b reduction will depend upon the concentration of E. PQH 2 or E ' DQH 2 (i.e., on the occupancy of sites by potential substrates) and the catalytic rate of oxidation of DQH 2 and PQH 2 by the enzyme. We assume that DQH 2 and PQH 2 can he displaced by DQ. Therefore, the initial rate of cyt b reduction will determined by the binding constants (from the lipid phase) for DQH 2, PQH 2 and DQ and by their concentrations in the lipid phase. The later will be determined by the partition coefficients for each species into the membrane and the concentrations added (the concentration of P Q H 2 was assumed from literature values, see text). In the scheme, PDQH2 and PDQ represent the partition coefficients of DQH 2 and DQ from the aqueous to the lipid phases, respectively - that for PQH 2 is considered to be infinite (i.e., all PQH 2 will be in the lipid phase), k e and k D represent the rate constants for the catalytic events with PQH 2 and DQH 2 bound at the Qo site, respectively and Kin,p, Km,o and K I are the disassociation coefficients for binding of PQH 2, DQH 2 and DQ, respectively, from the lipid phase to the Qo site. The state denoted 'P' refers to product, in this case reduced cyt b. See text for additional assumptions and conditions.

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strate, rather than the binding of substrate. This assumption does not rule out the binding of substrate or the unbinding of product (PQ or DQ) as rate limiting steps under steady-state conditions; it merely implies that all binding reactions will have reached equilibrium during the extended dark adaptation time (3 min), before the single-turnover actinic flash. We expect the oxidation of the high potential chain components to proceed at a significantly higher rate than the turnover at the Qo site (see Refs. [17,18] and above). Therefore, we expect the initial rate to predominantly reflect the catalytic rate of oxidation of bound substrate and the fraction of complexes occupied by substrate. We assumed that the binding constants for all species to the Qo site did not change with experimental conditions. We also assumed that the equilibrium constant for the formation of product was sufficiently high that, during the measurement of initial rate (for a period of 2 ms after the actinic flash), the rate of the back reaction would not have been significant. This latter assumption was perhaps the most susceptible to error since, with the addition of NQNO, the reaction is particularly susceptible to product inhibition (e.g. by the formation of reduced cyt b) and because the forward reaction the oxidation of P Q H 2 by oxidized cyt b and cyt f in a concerted reaction - has an equilibrium constant of only about 10 [25]. Despite this, we feel that the assumption is reasonable since we were always able to fit the initial phase of cyt b reduction with a reasonably straight line through the origin and through data point up to 2 ms after the actinic flash (see above). The rate equation for the simulations was derived as in Segel [26] for an enzymatically catalyzed reaction with two alternate substrates, but modified to include a competitive inhibitor. Briefly, the instantaneous rate of turnover of the enzyme is proportional to the fraction of catalytic sites occupied by a substrate multiplied by the Vmax for that substrate (the rate when all catalytic sites are occupied by that substrate). In the case with two competing substrates, the rate of turnover is proportional to the sum of the fractional occupancies multiplied by the Vma~ values for each substrate respectively. When a competitive inhibitor is included, the total enzyme concentration includes a term for the fraction of enzyme with the inhibitor bound. Thus, using the terms for states and equilibrium and rate constants shown in Scheme 1, the rate of turnover is given by v = kp'[E'PQH2] + k D.[E.DQH2]

(1)

where v is the instantaneous rate of turnover and kp and k D are the rate constants for the catalytic events with P Q H 2 and D Q H 2 bound at the catalytic site, respectively, E represents the enzyme (bf complex), E . P Q H 2 and E . D Q H 2 represent the enzyme with P Q H 2 and D Q H 2 bound at the Qo site, respectively.

The concentrations of the enzyme-substrate complexes can be expressed in terms of disassociation, since [E]total = [E] + [E'PQH2] + [E.DQH2] + [E'DQ]

(2)

where [E]total is the concentration of free and bound enzyme, and E . DQ represents the enzyme with DQ bound at the Qo site. Then, Km,p

[E]. [PQH 2] [E.PQH2]

(3)

Km,o

[E]'[DQH2] [E.DQH2]

(4)

[E].[DQ] [E.DQ]

KI

(5)

where Km,p, Kin,D and K I are the disassociation coefficients for binding of PQH2, D Q H 2 and DQ, respectively, from the lipid phase to the Qo site. It follows that, u

[E]total kp

=

[E]÷

[E]'[PQH2]

Km,p [E]'[POH2] Km,p

+

+k o

[E]'[DQH2]

Km,D [E]'[DOH2] [E]'IDO] Km,D

+- -

(6)

KI

Since Vmax is defined as the rate of turnover where all catalytic sites are occupied by the substrate, Vmax,p = kp[E]total Vmax,D = kD[E]tota,

(7) (8)

where Vmax,P and Vm~,,o are the maximal rates of turnover of the complex by P Q H 2 and DQH2, respectively. Then, by substitution, Vmax,p[PQH2 ] [-Vmax.D[DQH] Km,p Km,D V= [poll2] [DQH2] [DO] (9) 1+-[---+-Km,p

Km,D

KI

One assumption for applying this equation is that the concentrations of the free substrates or the inhibitor will not be significantly diminished by their binding to the catalytic site. This is not a problem with most treatments of enzyme systems because of the usual large excess of substrate over enzyme. Though the membrane phase concentrations of D Q and D Q H 2 are expected to be small in some cases, they are expected to be in equilibrium with a relatively large pool in the aqueous phase, allowing the concentration of free D Q and D Q H 2 in the membrane to remain essentially constant. However, P Q H 2 is expected to be exclusively partitioned into the membrane phase at a concentration only 6-fold over that of the catalytic site. This means that, given a constant total (free and bound) concentration of P Q H 2 in the membrane of 1 mM (see Refs. [27] and below), the concentration of free P Q H 2 would be 0.83 mM when 1 P Q H z was bound at each Qo site. On the other hand, this error is probably small

D.M. Kramer et al. / Biochimica et Biophysica Acta 1184 (1994) 251-262

compared to the relatively large degrees of freedom in the estimation of some of the other parameters. 4.4. Competition at the Qo site among P Q H 2, D Q H 2 and DQ The curves in Fig. 3 show a best global fit of the above rate equation to the entire data set in Fig. 3, using the computer program described in Materials and methods. The free-fitting parameters used in the simulation shown in Fig. 3 were the disassociation constants for PQH 2, D Q H 2 and DQ into the Qo site. The remaining parameters were assumed from literature sources or measured in our preparation. Since we expected no significant concentration of PQ (see above), it was not included in the simulation. The concentrations of DQ and D Q H z in the lipid will depend on the amount of the species added to the suspension, the partition coefficient for that species from the aqueous phase into the membrane and the relative volumes of the lipid and aqueous phases. The concentration of PQ in the membrane was taken from the estimate of Rich [27] and from this value, the ratio of cyt f: PS I : P S II (about 1: 1: 1, see above), the concentration of cyt f in our preparation (see above) and our measured ratio of P Q : P S I or PS II (about 5 - 6 : 1 , see above), we estimated the ratio of lipid to aqueous phases to be approx. 320 /xl/l of suspension medium. For partitioning of DQ and D Q H 2 into the thylakoid membrane, we used values obtained by Rich and Harper [28] for partitioning of these species from water into model organic phases. For partitioning from water to cyclohexane, these authors found a large difference in the partition of the DQ and DQH 2. The quinone form strongly partitioned into the organic phase, but the quinol form partitioned more favorably into the aqueous phase. On the other hand, much less difference was observed between the partitioning of the quinone and quinol forms when octanol was used as the organic phase. This is explained by the fact that octanol can provide hydrogen bonds to the hydroxyl groups of the quinol form. For the fit in Fig. 3, the octanol/water values were used (P(duroquinone)= 331; P(duroquinol) = 63.1), though similar fits could be obtained with the cyclohexane/water value (see below). When simulations were run, we found that a range of values gave adequate fits to the data, as long as the ratio of predicted disassociation constants was gm. P : gm, D : K~ = 1 : 0.34 : 0.39. In order to determine the extents of this range, we set the value of the binding constant for PQH 2 to a range of values and allowed the remaining binding constants to be free fitting parameters. The rms error of the best fit to the data as a function of the set value of Kin,P (the disassociation constant for PQH z) is plotted in the inset to Fig. 3. To adequately fit the data, the value of Km,1,

259

was restricted to less than about 5 • 10 -5 M (the disassociate constant). In other words, to fit our data with the above model, we had to assume a binding constant for PQH z to the Qo site greater than about 2.104 M-~ (the association constant). A similar value for the predicted binding constant for PQH 2 to the Qo site was obtained using the partition coefficients for both octanol : water and cyclohexane : water systems. A relatively tight binding for DQH 2 and DQ is predicted by the simulations, though the exact values depend greatly on the partition coefficients used in the simulation. If the partition coefficients into cyclohexane were used, the concentration of DQH 2 in the lipid phase was expected to be quite low - about 1% of that expected from the octanol:water system - and consequently, the binding constant predicted by the simulation became roughly proportionally stronger; the ratio of disassociation constants became 1:0.003:0.5, for K m , p : K m , D : K I. In this case a large difference between the binding of quinone and quinol forms to the Qo site would imply a significantly higher midpoint potential for the D Q / D Q H 2 couple, bound at the Qo site, compared to the same couple in the membrane. If we assume that the thylakoid membrane is similar to the cyclohexane :water system, the large difference in partitioning of the quinol and quinone form found in the membrane would also imply a dramatic shift in the midpoint potential of the D Q : D Q H 2 couple in the membrane from that found in the aqueous phase. If the difference in partitioning between the quinone and quinol forms also holds for the P Q / P Q H 2 couple, it follows that the midpoint potential of the PQ pool would be expected to be lowered by as much as about 100 mV from that of P Q : P Q H 2 dissolved in water. Rich and Harper [28] argue that, since the midpoint potential of the pool appears to be in the same range as that of more water soluble analogs (about 100-120 mV), it seems unlikely that a major shift in the midpoint potential is affected by the lipid environment. Therefore, they favor a microenvironment for the lipid phase closer to that modeled by octanol. In any case, without further experimentation, it is difficult to estimate the true partition coefficients for the quinone: quinol species, or to know if all species partition into the same microenvironment of the lipid bilayer-protein system. We treat the simulations obtained above as tentative until more reliable data on partitioning is available. Our conclusions about the binding of PQH 2 to the Qo site of the bf complex can be compared to the case of the bc I complex. Crofts and Wang [29] summarized extensive studies of the cytochrome bc 1 complex of Rhodobacter spheroides and concluded that the binding of ubiquinol (UQH 2) to the Qo site is moderately tight; with essentially all Qo sites occupied by ubiquinol (UQH 2) when the pool is reduced. This is similar to

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D.M. Krameret al./ Biochimica et BiophysicaActa 1184 (1994)251-262

the case of the upper limit of disassociation constants (i.e., lower limit of binding constants) predicted by the kinetic model (above), where essentially all Qo sites are expected to be occupied with a fully reduced PQ pool. It should be emphasized, however, that, because the concentration of UQH 2 in the chromatophor membrane is expected to be 15-30-fold higher than the concentration of PQ in the thylakoid membrane (see Ref. [29] and references within), the binding of PQH 2 to the Qo of the bf complex is likely to be at least this much tighter than the binding of UQH 2 to the same site of the bc 1 complex. At this time, our data does not allow us to draw any conclusions about the binding of PQ to the Qo site. 4.5. Interaction of other quinone : quinol couples with the Qo site We made preliminary attempts to characterize the interaction of other quinone:quinol couples with the bf complex and briefly summarize these findings below (data not shown). Even at low concentrations (lower than 10 /~M), decyl-ubiquinol (dUQH2) , added to anaerobic samples, gave nearly maximal rates of cyt b reduction upon flash excitation. With 20/zM dUQH2, addition of up to 50/zM DQ, which markedly affected the binding of DQH 2 and PQH 2 (see above) to the Qo site, slowed the initial rate of cyt b reduction by only about 20%, indicating that dUQH 2 acts as a substrate for the complex and effectively out-competes duroquinone for the Qo site. However, addition of low concentrations (1-10/~M) of dUQ resulted in a marked slowing of cyt b reduction, indicating that dUQ competes with dUQH 2 for the Qo site. Decyl-ubiquinone and dUQH 2 are expected to partition more strongly into the lipid phase than DQ or DQH 2 [28], but since the partition coefficients are not known, it is not possible to determine whether the apparently high activities of dUQH 2 and dUQ at the Qo site are due simply to stronger partitioning into the lipid phase, or to a combination of stronger partition coefficients and increased affinities at the Qo site. Trimethyl-p-benzoquinone (TMQ) and its reduced form (TMQH 2) gave results similar to those obtained with DQ and DQH 2. Anaerobic thylakoid suspensions, prepared as in the experiments of Fig. 2 and reduced with 100/zM TMQH 2 showed an initial rate of cyt b reduction following a flash, of about 75% of the maximal rate. Addition of 10 ~ M TMQ to these samples slowed the initial rate to about 40% of maximum. Since the midpoint potential of the couple ( E r a , 7 = + 114 mV, see Ref. [16]) is near that of the PQ pool, it was difficult to determine what fraction of the effects were due to addition of a competitive inhibitor or to titration of the PQ pool. However, in light of the fact that the initial rate of cyt b reduction by the PQH 2

was not slowed significantly by oxidation of the PQ pool by 20% (see above), it is clear that, the addition of about 10% oxidized TMQ had effects beyond its effect on the redox poise of the PQ pool. This result is consistent with a competition of the quinone form for the Qo site. 4.6. Relationship between the rate of turnover of the Qo site and that at the Qi site: dependence of the cyt b oxidation kinetics on competition among quinone and quinol species It has been previously observed that the kinetics of re-oxidation of cyt b appeared to be related to the redox state of the plastoquinone pool. Under oxidizing conditions (e.g. with addition of 100-500 /zM sodium ferricyanide, see Joliot and Joliot [10]) re-oxidation of cyt b appears to be very slow (tl/2 of about 800 ms). On the other hand, under conditions where the PQ pool is reduced, re-oxidation of cyt b competes kinetically with its reduction so that the net reduction of cyt b during a trace is very small. Intermediate conditions produce intermediate kinetics [8, 9]. It was concluded that the rate of oxidation of cyt b is dependent on the redox poise of the PQ pool and several models have been proposed to explain this dependence (see Joliot and Joliot [30] and references within). The data in Figs. 1 and 2 show that, even in the presence of NQNO, the rate of re-oxidation of cyt b is more rapid with higher concentrations of DQH 2 and consequently lower ambient redox poises. However, the oxidation rate does not appear to depend simply on the ambient redox potential of the PQ pool. This is illustrated in Fig. 1, where the rate of re-oxidation of cyt b is more rapid in pre-illuminated samples after one flash (fully reduced PQ pool) and after a second (approx. 20% oxidized PQ pool - at a redox poise for the PQ pool of approx. + 100 mV), than at significantly lower redox poises in the presence of DQH 2 and DQ (e.g. Fig. 2, trace B, where the poise should be approx. +60 mV). Indeed, the re-oxidation kinetics appear, instead, to parallel the reduction kinetics regardless of the ambient potential. It seems reasonable to suggest that the variation in re-oxidation kinetics is, as with the reduction kinetics, the result of competition among quinone/quinol species for a binding site. The similarity of dependencies of the reduction and oxidation kinetics on the concentrations of competing species suggests that either the same binding site or that two sites having very similar binding constants for the three competing species are in involved. This result is consistent with a model we have proposed to account for the slow re-oxidation kinetics of cyt b at high potential [31]. In this model, cyt b oxidation occurs by a two-electron process since the oxidant is a P Q : P Q H 2 couple at the Qi site with an

D.M. Kramer et al. / Biochimica et Biophysica Acta 1184 (1994) 251-262

unstable semiquinone intermediate. Multiple turnovers are prevented at high potential since the limiting substrate, P Q H 2 is not readily available. The oxidation of cyt b, then, is dependent on a second turnover of the Qo site. Excluding quinol species from the Qo site by addition of a competitive inhibitor, such as DQ, will slow not only the reduction, but also the re-oxidation of cyt b. The rate of cyt b re-oxidation will depend not solely on the redox state of the PQ pool but on the activity of quinol species at the Qo site.

4. 7. Implications for steady-state turnover of the bf complex It has been previously observed that the rate of turnover of the bf complex at the expense of D Q H 2 under steady-state conditions (Graan and Ort, personal communication) and upon closely-spaced actinic flashes does not saturate even at very high D Q H 2 concentrations (above 1 mM). This has been attributed by Rich et al. [5] to a slow transhydrogenase activity in thylakoids. At the same time, these authors argue that D Q H 2 is a substrate for the complex. In order to reconcile these two assertions, in the absence of additional hypothesis, it would be necessary to postulate that D Q H 2 reacts more slowly with the bf complex than does P Q H 2 - either because a step in the oxidation of D Q H 2 is slower than that of P Q H 2, or because the activity of D Q H 2 at the quinol oxidase site is lower than that of P Q H 2 (this may be attributed to a lower lipid phase concentration of DQH2, or to a lower binding of D Q H 2 to the Qo site, or both). However, we show that the Vmax for turnover by D Q H 2 is approximately equal to that by the native P Q H 2 and that a rate equal to Vma~ can be achieved at relatively low concentrations of D Q H 2, provided that D Q is not produced upon addition of D Q H 2. As we discussed above, it is clear that the slow turnover kinetics in the presence of D Q H 2 are directly related to the presence of a competitively binding inhibitor (DQ) that was most likely present in these previous studies in sufficient quantities to significantly slow the turnover of the complex. We conclude that our competitive binding model can be used to explain the majority of the data in these previous reports. These studies emphasize the possible pitfalls of using analogs of redox-active electron transport components in functional assays. We conclude that, under conditions used in most steady-state assays of the bf complex using D Q H 2 as electron donor (aerobic samples with a high concentration of electron donor), the vast majority of turnovers of the complex most likely occur at the expense of D Q H 2 , not P Q H 2 as has been previously suggested (e.g., see Ref. [32] and references within). Furthermore, these assays were most likely carried out in the presence of an inhibitor (DQ) which

261

was not accounted for. This implies that many studies of the bf complex have in fact explored a rather artificial system that is perhaps not directly comparable to the native system. We conclude that any extrapolation of results using artificial donors and acceptors to the native system must be viewed with caution, especially when the interaction of electron donor has not been thoroughly characterized and when the turnover of the native system without addition is not well characterized. On the other hand, studies of the interaction of artificial donors and acceptors may yield interesting information about the turnover of the complex and have in this work led to an estimate of the upper limit of the binding constant for P Q H 2 to the Qo site.

5. Acknowledgments The authors thank Dr. Peter Rich for interesting discussions and Dr. Vladimir Shinkarev for a critical reading of the manuscript. This work was supported by the U.S. Department of Energy ( D O E D E F 60286ER13594) and by a McKnight Foundation Award for Interdisciplinary Research.

6. References [1] Dutton, P.L. (1978) Methods Enzymol. LIV, 411-435. [2] White, C.C., Chain, R.K. and Malkin, R. (1978) Biochim. Biophys. Acta 502, 127-137. [3] Izawa, S. and Pan, R.L. (1978) Biochem. Biophys. Res. Comm. 83, 1171-1177. [4] Jones, R.W. and Whitmarsh, J. (1985) Photobiochem. Photobiophys. 9, 119-127. [5] Rich, P.R., Madgwick, S.A. and Moss, D.A. (1991) Biochim. Biophys. Acta 1058, 312-328. [6] Moss, D.A. and Bendall, D.S. (1984) Biochim. Biophys. Acta 767, 389-395. [7] Arnon, D.I. (1949) Plant Physiol. 24, 1-15. [8] Velthuys, B.R. (1978) Proc. Natl. Acad. Sci. USA 75, 6031-6034. [9] Velthuys, B.R. (1978) Proc. Natl. Acad. Sci. USA 76, 2765-2769. [10] Joliot, P. and Joliot, A. (1984) Biochim. Biophys. Acta 765, 210-218. [11] Rich, P.R. Heathcote, P. and Moss, D.A. (1987) Biochim. Biophys. Acta 892, 138-151. [12] Joliot, P., Beal, D. and Frilley, B. (1980) J. Chim. Phys. 77, 209-216. [13] Joliot, P. and Joliot, A. (1986) Biochim. Biophys. Acta 849, 2111-222. [14] Kramer, D.M. (1990) Ph.D. Thesis, University of Illinois. [15] Clark, W.M. (1972) Oxidation-Reduction Potentials of Organic Systems. Robert E. Krieger Publ. Co., Huntington, NY. [16] Swallow, A.J. (1982) in Function of Quinones in Energy Conserving Systems (B. Trumpower, ed.), pp. 59-72, Academic Press, Inc. [17] Bouges-Bocquet, B. (1977) Biochim. Biophys. Acta 462, 371-379. [18] Kramer, D.M. and Crofts, A.R. (1990) Photosynth. Res. 23, 231-240. [19] Graan, T. and Ort, D.R. (1984) J. Biol. Chem. 259, 14003-14010. [20] Lavergne, J. (1982) Photobiochem. Photobiophys. 3, 273-285.

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