7850
J. Phys. Chem. B 1997, 101, 7850-7857
Binding of Ubiquinone to Photosynthetic Reaction Centers: Determination of Enthalpy and Entropy Changes in Reverse Micelles Antonia Mallardi CNR, Centro Studi Chimico-Fisici sull’Interazione Luce-Materia, Via Orabona 4, I-70126 Bari, Italy
Gerardo Palazzo* Dipartimento di Chimica, UniVersita` di Bari, Via Orabona 4, I-70126 Bari, Italy
Giovanni Venturoli Laboratorio di Biochimica e Biofisica, Dipartimento di Biologia, UniVersita` Bologna, Via Irnerio 42, I-40126 Bologna, Italy ReceiVed: March 5, 1997; In Final Form: June 27, 1997X
The photosynthetic reaction center from the purple non-sulfur bacterium Rhodobacter sphaeroides has been solubilized in reverse micelles of phospholipids in n-hexane. The rate of the charge recombination from the secondary quinone acceptor (QB) to the bacteriochlorophyll dimer (P) has been investigated, by flash absorption spectroscopy, as a function of the concentration of ubiquinone-10 at eight temperatures between 37 and 6 °C. Deconvolution of P+ decay shows that, in reverse micellar solutions, quinone molecules are in fast exchange between the QB site of the protein and the bulk organic phase, with the exception of a small fraction of reaction centers lacking the QB functionality. Global analysis of the kinetics of charge recombination permits proper separation of the contribution of the binding at QB from that of the P+QA-QBf P+QAQB- electron transfer. Enthalpy and entropy changes obtained for the electron transfer process (∆HAB° ) -0.140 ( 0.016 meV; ∆SAB° ) -2.01 × 10-4 ( 6.7 × 10-5 meV/K) are in agreement with previous evaluations in detergent suspensions of reaction centers. Thermodynamic parameters of the enthalpically driven quinone binding at QB (∆Hbind° ) -0.526 ( 0.058 meV; ∆Sbind° ) -1.36 × 10-3 ( 2.3 × 10-4 meV/K) compare favorably with data in aqueous systems when considering a free energy change of about -4.77 kJ/mol for the transfer of ubiquinone-10 from a direct detergent micellar phase to a n-hexane reverse micellar solution. This comparison indicates that the quinone affinity for the QB site is approximately the same in the (dark) PQA and (light) P+QA- states of the reaction center.
Introduction In photosynthetic and respiratory electron transfer chains, the quinones play a relevant role as mediators of vectorial electron and proton transport. The quinone molecules are localized within the biological membrane, where they form a freely diffusing “pool” component. However, the electron transfer reactions and the coupled proton binding and release processes involving quinones take place at specific binding sites localized in specific membrane redox protein complexes. The comprehension of the different equilibrium of binding and of the electron transfer features at the quinone catalytic sites is the key to obtaining insight into the mechanism of the efficient capture and storage of free energy in photosynthesis and respiration. In photosynthetic reaction centers (RCs), proteins that extend across the chloroplast membrane of green plants1 and the intracytoplasmic membrane of photosynthetic bacteria,2 absorption of light energy gives rise to a series of single electron transfer reactions involving quinones, which result in a charge separation across the membrane. In the purple non-sulfur bacterium Rhodobacter sphaeroides, the charge separation involves the transfer of an electron from a bacteriochlorophyll dimer (the primary donor, P) to the primary ubiquinone acceptor * Author to whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, August 15, 1997.
S1089-5647(97)00825-0 CCC: $14.00
(QA).2 Electron transfer normally proceeds from QA- to the secondary ubiquinone (QB). After re-reduction of the primary donor by cytochrome (cyt) c2, a second turnover of the RC leads to reduction of the semiquinone molecule at the QB site. Following binding of two protons, the resulting ubiquinol (QH2 ) leaves the protein and rapidly exchanges with the pool of ubiquinone (Q) molecules in the hydrophobic part of the membrane.3 A second integral membrane complex, the cyt bc1 complex, oxidizes QH2 molecules and transfers electrons to cyt c2 to complete the light-driven electron cycle. In the RC, ubiquinones bound at the QA and QB sites display distinct properties: at the QA site quinone operates as a nonexchangeable cofactor accepting a single electron, while the quinone bound at the QB site can be fully reduced and protonated and exchanges with the membrane quinone pool.2 In the bacterial RC the role of the protein in the binding and in the electron transfer properties of the quinones at the QA and QB sites has been investigated in detail, taking advantage of the structures of the protein and cofactors known at atomic resolution.4,5 In contrast, little is known on the physicochemical features of the overall binding processes. The tendency to overshadow the study of the overall binding process is in part due to the extreme hydrophobicity of the integral membrane protein and of the native quinones, which can be solubilized in aqueous systems only by substitution of the membrane phospholipids with detergent molecules. In these microheterogeneous systems both the equilibrium and the © 1997 American Chemical Society
Binding of Ubiquinone to Photosynthetic RCs kinetics of quinone binding are strongly influenced by the structure and dynamics of the surfactant aggregates.6 In the presence of a topologically disconnected lipophilic domain and of a continuous aqueous bulk, assumptions on the quinone activity coefficients must be made,6 and a distinction between the contribution of solvent water and direct protein-quinone interaction to the binding of these hydrophobic ligands is required.7,8 For these reasons the present investigation has been performed in reverse micellar solutions of phospholipids in n-hexane in order to have a system characterized by a continuous lipophilic bulk where the strong interfering contributions of quinone aqueous desolvation effects are absent.7,8 Phospholipids in apolar, organic solvents spontaneously form micelles. Such reverse (or inverted) micellar aggregates can be schematically described as consisting of water droplets, surrounded by the polar heads of the phospholipid molecules facing into the interior of the micelle, while the hydrophobic chains are directed toward the bulk, continuous organic phase.9 Although the existence and the possible role played by such structures in native membranes remain a topic of research and discussion, reverse micelle solutions provide a powerful tool in investigating, in Vitro, the interaction of integral membrane proteins with hydrophobic cofactors. The anisotropic and amphipatic nature of biological membranes is preserved in these organized microassemblies, as well as the functionally relevant interface between phospholipid head groups and water. More importantly, the presence of a continuous organic phase, in which hydrophobic compounds are soluble, allows a detailed study of the interaction of liposoluble molecules with membrane proteins, avoiding possible artifacts present in aqueous solutions (direct micelles of detergent and liposomes). The present study deals with the determination of the thermodynamic parameters (namely enthalpy and entropy changes) of the binding of ubiquinone to the QB site of the RC from Rb. sphaeroides. Materials and Methods Chemicals. Ubiquinone-10, phosphatidylserine (brain Extract, type III: Folch fraction III from bovine brain) and phosphatidylethanolamine (type II-S: from sheep brain) from SIGMA were used without further purification. Soybean phosphatidylcholine (Epikuron 200) was a generous gift from Lucas-Meyer. Sephadex G-50 was purchased from Pharmacia and terbutryne from Chem Service. n-Hexane (for UV spectroscopy) and lauryl dimethylamine oxide (LDAO) were from Fluka. Reaction Centers. RCs were isolated and purified from Rb. sphaeroides R-26 as already described.10 In all preparations the ratio between the absorption at 280 nm and the absorption at 800 nm was about 1.2. The concentration of the RC was determined spectrophotometrically by the absorbance at 802 nm (802 ) 288 mM-1 cm-1). The RC was stored in TLE buffer (10 mM Tris-HCl, 0.025% LDAO, and 1 mM EDTA, pH) 7). Preparation of RC-Containing Reverse Micelles. A phospholipid mixture of phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) at a ratio of 2:1:1 was dispersed in 10 mM imidazole, 100 mM KCl, and 3% Na-cholate buffer, pH ) 7, and sonicated on ice for 3 min. The RC was added to the suspension at approximately 30 µM of final concentration, in order to obtain a molar ratio total lipids/ RC of 4500/1. The final mixture (0.5 mL) was sonicated twice for 3 s and then applied onto a column (1 × 10 cm) of Sephadex G-50 equilibrated in 10 mM imidazole and 100 mM KCl buffer, pH ) 7. The fraction containing the reconstituted proteoliposomes was diluted to 2 mL and extracted with 200 µL of 1 M
J. Phys. Chem. B, Vol. 101, No. 39, 1997 7851 MgCl2 and 3 mL of n-hexane with a vortex mixer for 3 min at room temperature. The two phases were separated by centrifugation for 5 min in a desk centrifuge, and the organic phase was retained. Usually, the RC-containing reverse micelles obtained from different preparations were pooled in order to minimize heterogeneity due to the extraction procedure. Samples of 1 mL each were taken from the pool and used for the kinetic measurements at a given temperature varying the quinone concentration. Ubiquinone-10 (Q) was added to the sample at the desired concentration as n-hexane solution; the uncertainty in the quinone concentration was less than 5%. In RM the RCs retain their photoactivity for several days, if stored at 4 °C in the dark.11,12 The absorption and circular dichroism spectra of RC solubilized in this reverse micellar system reveal the same spectral features of RC in aqueous LDAO solutions, as reported by several authors.7,11-13 Due to the high affinity of the ubiquinone for the n-hexane, both the native quinones (QA and QB) are removed in this system.11,12 The occupancy of the quinone binding site (and thus the photoactivity of the protein) can be restored dissolving an excess of quinone in the organic bulk.11,12 Using such a procedure, the binding constant at 25 °C of ubiquinone-10 to the QA site has been recently evaluated.8 The solubility of ubiquinone-10 in n-hexane was determined by adding an excess of solid quinone to the hexane. After the mixture was stirred for several hours at 298 K, the solid was removed by centrifugation and the quinone concentration in the supernatant was determinated spectrophotometrically by the absorbance at 403 nm (403) 554 M-1 cm-1). The quinone solubility in LDAO was determined as follows. Ubiquinone10 in ethanol was evaporated, and 1% LDAO in 10 mM imidazole and 100 mM KCl buffer was added. The mixture was stirred at 298 K for several hours, and the undissolved quinone was removed by centrifugation. The concentration of ubiquinone was determined by measuring the change in optical absorption at 275 nm after reduction by NaBH4. Spectroscopic Measurements. The absorption spectra of RCs were measured in a 1 cm path length cell using a doublebeam Jasco 8700 spectrophotometer. Flash-induced redox changes of the primary donor of the RC solubilized in reverse micelles were monitored at 600 nm with a single-beam spectrophotometer of local design, using a 1 cm path length cell closed by a conical Teflon cap in order to prevent the solvent evaporation. Actinic flashes were provided by a xenon lamp (3.25 J discharge energy), screened through two layers of Wratten 88A gelatin filter, giving a light pulse of 4 µs duration at half-maximal intensity. The photomultiplier (PMT) was protected by a Corning L46 glass filter. By means of a beam splitter placed at the entrance of the sample holder, a portion (10%) of the incident light was fed to a photodiode and its signal used to correct for fluctuation in the intensity of the measuring beam over the hundreds of milliseconds time range. Rapid digitization and averaging of the amplified PMT signal was done by a Le Croy TR 8818 transient recorder equipped with a 128 K memory module (MM8105) and controlled by an Olivetti M290 personal computer. The cell holder was thermostated using a cryothermostat (Haake F3K). The temperature of the sample was monitored by a Pt-100 ceramic resistance (Degussa GR 2105) with a tolerance of 0.3 °C, immersed directly into the sample. The sequential analysis into multiple exponential decays and the simultaneous global analysis described in the text were performed by using the “STEFIT” program (STELAR s.n.c.). The program allows the data to be fit by routines based on three
7852 J. Phys. Chem. B, Vol. 101, No. 39, 1997
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different algoritms (Simplex,14,15 Powell,15 and Quadric15), limiting the risk of relative minima in the least-squares norm. Confidence limits were evaluated for all estimated parameters at 95% confidence as linear joint confidence intervals.15,16 The Hessian matrix that is required for this procedure was evaluated by the method outlined in the appendix to ref 14. Results In reverse micelles, the RC lacks any exogenous electron donor to P+ and the charge recombination between the flashgenerated species, P+ and QB-, is observed. If, otherwise, the QB binding site is empty or the electron transfer from QA- to QB is blocked, the light-induced charge separation and the following charge recombination are limited to the P+QA- state. Since the negative charge is more stabilized when it resides on QB, charge recombination from P+QA- is faster than that from P+QB-.2 Consequently, the observed kinetics of P+ decay depend on the parameters governing the quinone exchange.17 We have investigated the dependence of the kinetics of charge recombination on the quinone concentration at eight different temperatures between 37 and 6 °C. For each temperature the decay of P+ was monitored at 600 nm as a function of the quinone concentration. Under the condition tested, the decay can be accounted for by two exponential phases. The results of this deconvolution procedure are summarized in Figure 1. Figure 1A shows the quinone dependence of the recombination kinetics at different temperatures (for sake of clarity only three temperatures are shown). The lifetime of the slow phase (τS) strongly depends on the quinone concentration ([Q]), increasing at increasing [Q]. This effect is observed over the whole temperature range. Furthermore, at fixed [Q], a decrease in temperature results in an increase in τS, as already reported.18 On the other hand, the lifetime of the fast phase (τF) does not show any correlation with both the quinone concentration and the temperature. The τF values obtained following this deconvolution are not constant but fluctuate along the isothermal experiment and do not reveal any trend when the temperature is changed. More important, the relative amplitude of the fast phase (PF) remains constant within the experimental error, for each binding isotherm (Figure 1B), and in general seems to be a characteristic of the extraction procedure (samples from the same pool of n-hexane extracts show about the same PF; data not shown). At 37 °C the absorption spectrum of the RC solubilized in reverse micelles reveals an increase in the ratio A760/A865, which raises up to 1.68 (at lower temperatures A760/A865 ≈ 1.3). This feature reflects the degradation of a fraction of the proteins;11 the traces recorded following a light flash at this temperature are more noisy but still follow the trend above outlined. In Rb. sphaeroides RCs the charge recombination from the P+QB- occurs only by thermal repopulation of the P+QAstate.19 As a consequence, the decay of P+ is slower in RCs with the QB site occupied because the electron transfer to QB diminishes the time-averaged population of QA- (the electron transfer time (kAB + kBA)-1 is fast compared to the recombination time kAP-1). As it is discussed in detail in the literature, the observable kinetics of the P+QB- recombination depend, in principle, on the time scale of both the preflash and the postflash binding of the quinone to the QB binding site in accordance with the scheme of Figure 2. Under conditions of high quinone concentration, when a pseudo-first-order approximation holds for the binding at QB site, the kinetics of P+ decay, following a flash, can be derived analytically from the scheme of Figure 2 (for comprehensive review see ref 17).
Figure 1. [Q] dependence of P+ relaxation in reverse micelles at three temperatures: results come from a two-exponential deconvolution of each decay. (A) Life times of the slow (filled symbols) and fast (open symbols) components. (B) Relative amplitude (PF) of the fast phase.
In analyzing the kinetics of P+ dark recovery in terms of the scheme of Figure 2, it is useful to consider three cases separately, i.e.: (a) the QB binding equilibrium is slow compared to the rate, kAP, of P+QA- charge recombination (kAP ≈ 10 s-1 in Rb. sphaeroides RCs2,20); (b) the rates of exchange of quinones are of the same order of magnitude of kAP; (c) a fast exchange occurs at the QB site. The kinetic behaviors expected in cases a and b are incompatible with the results obtained by us in n-hexane. For a slow exchange, biphasic kinetics of P+ recovery are predicted, with rate constants independent of the quinone concentration and with the fraction of the fast phase which decreases increasing [Q], in contrast with the data of Figure 1 in which τS increases strongly with the amount of quinone, whereas PF is essentially unchanged. In case b, P+ dark relaxation can still be described by biphasic exponential kinetics, with amplitude and rates depending upon the concentration of quinone, [Q]. It can be shown21 that both the sums and the products of the rate constants should increase with the quinone concentration. In the case of RC in n-hexane it happens just the opposite, τF being almost constant and τS increasing when [Q] is raised.
Binding of Ubiquinone to Photosynthetic RCs
J. Phys. Chem. B, Vol. 101, No. 39, 1997 7853
Figure 2. Kinetic relationships among the photoinduced charge separation, relaxation, and the quinone exchange at the QB site of the RC. kAP is the rate constant for electron transfer from QA- to P+; kAB and kBA are the forward and backward rate constants of electron transfer between QA and QB and define LAB ) kAB/kBA, the equilibrium constant for one-electron transfer from QA to QB. kQB and kBQ are, respectively, the binding and release rate constants of pool quinones at the QB site of the RC in the P+QA- state (“light” state). kQB* and kBQ* indicate the binding and release rate constants of pool quinones at the QB site of the RC in the PQA state (“dark” state). Accordingly, it is possible to define the equilibrium binding constant in the “light” (Kbind ) kQB/kBQ) and the “dark” (Kbind* ) kQB*/kBQ*) states.
Finally, when the quinone exchange is fast (case c), one can assume quasi-equilibrium between states P+QA- and P+(QAQB)during dark relaxation. The kinetics of P+ recovery can be described by a single-exponential decay with an effective rate constant kP determined by
kp )
kAP app 1 + LAB
(1)
where Lapp AB is the apparent equilibrium constant for the electron transfer between QA and QB and depends on the quinone concentration and on the equilibrium constant for the binding of quinones (Kbind) according to app LAB )
LABKbind[Q] 1 + Kbind[Q]
(2)
Kbind ) kQB/kBQ (see Figure 2) defines the binding to P+QAstate. Lapp AB is lower than the intrinsic equilibrium constant LAB of the electron transfer between QA and QB but approaches it at high quinone concentrations. As a consequence, the observed lifetime (kP-1) rises with quinone concentration up to a plateau. This behavior is compatible with the kinetics of the slow phase dominating the P+ decay observed by us in n-hexane; in fact at all the temperatures examined τS increases at increasing quinone concentration. We attribute the additional fast phase to a fraction of RCs in which the QB site functionality has been completely lost during the extraction in organic solvent. In this hypothesis the overall experimental P+ relaxation will consist of the contribution of RCs damaged at the QB site (with a rate constant kAP) and of a contribution due to the fully competent RCs (with a rate constant kP that is quinone dependent according to eqs 1 and 2). The independence of τF from the quinone concentration and the fact that experiments performed on samples coming from the same n-hexane extraction reveal variation on PF less than 10% strongly support our interpretation. To further test this hypothesis and to extract more reliable values for the kinetic parameters of the model, we have
Figure 3. Comparison between the experimental charge recombination kinetics and the global analysis (see text). The points represent the experimental values of absorbance change measured at different times after a light flash for solutions of the RC in reverse micellar solutions at different ubiquinone-10 concentrations. The lines represent the best global fit according to eq 3. Conditions: (A) T ) 6 °C, [RC] ) 1.7 µM; the continuous lines correspond to PF ) 0.119 (τF and the values of τS at different [Q] are listed in Table 1. (B) T ) 27 °C, [RC] ) 1.7 µM, the lines correspond to PF ) 0.095 (τF and the values of τS at different [Q] are listed in Table 1).
performed a global analysis22 of each subset of data taken at a given temperature (in the same sample), fitting the P+ decay to
P+(t) ) A0‚(PF‚e-t/τF + (1 - PF)‚e-t/τS)
(3)
where the same parameters PF (the fraction of fast phase) and τF (the lifetime of the fast phase) have been assumed for all the traces at a given temperature, while τS, the lifetime of the slow phase, is a function of [Q]. The parameter A0 is the extent of the bleaching at t ) 0 and acts as a free normalization constant in each signal. The results of this global analysis are shown in Figure 3 for two temperatures. The agreement between experimental data and fits is still good under the restrictive hypothesis of eq 3. The values of τF and τS for all the temperatures and [Q] investigated are listed in Table 1. The corresponding values of the fraction of fast phase, PF, obtained from global analysis of samples reveal a variability which appears to be mainly related to the aging of the samples. Within one day aging (i.e. time between extraction and measurements) the relative contribution of the fast phase never exceeded 10% of the total. PF increased up to 30% following a 4 days aging. This factor, however, does not affect our analysis of binding at QB, since even 4 days old samples have essentially the same values of τS of fresh n-hexane extract at a given temperature and [Q]. Since, according to the proposed interpretation, τF should coincide with τAP ) kAP-1, we have recorded the kinetics of P+ decay in the presence of excess (1.5 mM) terbutryne (TBT, an inhibitor of the QA-QB f QAQBelectron transfer) at all temperatures and at [Q] ) 13 mM. The average lifetime of the recombination from QA- in n-hexane obtained by fitting to one exponential plus a constant term23 was found to be τAP ) 110 ( 19 ms, in reasonable agreement with the τF values of Table 1. The value of the constant is around 2% of the maximum bleaching. Discussion For each temperature, the experimental P+ relaxation measured at different [Q] can be accounted for by two exponential phases. The ratio of the amplitudes of the two phases is independent of temperature and of quinone concentration for a given pool of n-hexane extract and within approximately 2 days of the sample aging. The values of τF obtained from the global analysis (Table 1) are in agreement with the average lifetime of the P+QA- recombination obtained from the single-
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TABLE 1: Lifetimes of the Fast Phase (τF) and the Slow Phase (τS) for Two-Exponential Deconvolution of P+ Decaya T ) 6 °C, τF ) 107 ( 39
T ) 11 °C, τF ) 80 ( 11
T ) 15 °C, τF ) 96 ( 23
T ) 19 °C, τF ) 97 ( 31
[Q] (mM)
τS (ms)
[Q] (mM)
τS (ms)
[Q] (mM)
τS (ms)
[Q] (mM)
τS (ms)
0.78 1.5 3.0 5.2 7.2 9.1 11.5 14.2
1289 ( 47 1875 ( 58 2351 ( 74 3024 ( 100 3149 ( 103 3452 ( 172 3309 ( 169 3584 ( 199
0.35 0.70 1.4 2.8 4.3 5.9 7.3 8.7 10.3 12.8
474 ( 33 701 ( 35 1045 ( 48 1545 ( 77 1914 ( 107 2229 ( 106 2389 ( 102 2358 ( 106 2686 ( 128 2657 ( 123
0.40 0.80 1.6 3.1 4.9 6.6 8.3 9.8 11.6 14.5
362 ( 20 542 ( 30 812 ( 51 1237 ( 67 1574 ( 96 1780 ( 91 2038 ( 105 2116 ( 115 2222 ( 119 2235 ( 126
0.80 1.6 3.1 4.9 6.6 8.3 9.8 12.6
440 ( 18 667 ( 29 1015 ( 42 1351 ( 55 1608 ( 60 1803 ( 66 1943 ( 73 2165 ( 82 (
T ) 23 °C, τF ) 171 ( 47
T ) 27 °C, τF ) 112 ( 40
T ) 32 °C, τF ) 145 ( 52
T ) 37 °C, τF ) 134 ( 85
[Q] (mM)
τS (ms)
[Q] (mM)
τS (ms)
[Q] (mM)
τS (ms)
[Q] (mM)
τS (ms)
0.80 1.6 3.1 4.9 6.6 8.3 9.8 11.6 13.3 15.0
367 ( 59 553 ( 24 888 ( 49 1188 ( 48 1289 ( 57 1475 ( 65 1537 ( 67 1728 ( 80 1836 ( 89 1868 ( 92
0.70 1.4 2.7 4.6 6.5 8.2 9.8 12.3
269 ( 14 412 ( 17 632 ( 27 920 ( 35 1070 ( 42 1218 ( 51 1379 ( 54 1468 ( 60
0.74 1.4 2.9 4.9 6.8 8.6 11.1 13.9
206 ( 16 313 ( 18 488 ( 26 676 ( 32 898 ( 42 1010 ( 48 1117 ( 54 1275 ( 69
0.80 1.56 3.1 5.3 7.3 10.5 13.4
150 ( 23 243 ( 25 419 ( 38 532 ( 44 640 ( 56 891 ( 70 1075 ( 60
a
For all the [Q] at a given temperature (same sample), the same τF was assumed according to eq 6 (see text for further details).
exponential deconvolution of the P+ decay observed in n-hexane in the presence of TBT (see Results) and close to the values found in aqueous systems20 in the same range of temperature. These features show that in a minor fraction of RCs the electron transfer from QA- to QB is inhibited. Recent studies performed on a similar system (organogels) indicate a marked influence of the ratio water/lecithin on the functionality of the QB site.24 For all the temperatures the value of the lifetime of the slow component (τS) obtained from global analysis was found to increase at increasing [Q] up to a plateau. As a whole these results confirm that in reverse micellar solutions quinone molecules are in fast exchange between the QB sites of RCs and the organic phase, with the exception of a small fraction of RCs lacking the QB site functionality. Accordingly, the dependence of the lifetimes of the slow phase on the quinone concentration can be related to the values of the electron transfer (LAB) and binding (Kbind) constants at each temperature. Equation 1 can be rewritten in terms of lifetimes giving app τP ) τAP(1 + LAB )
(4)
where τP is the lifetime of charge recombination in the fully competent RC and correspond to τS of Table 1; τAP is the lifetime of the charge recombination from QA- and can be obtained from the experiments in presence of TBT. In principle, eq 4 would allow evaluation of Lapp AB for each temperature and [Q]. In turn, at a fixed temperature, Lapp AB is a function of LAB, Kbind, and [Q] according to eq 2. Unfortunately the uncertainties in the Lapp AB values are not negligible (τP and τAP come from a deconvolution procedure). Furthermore LAB and Kbind are strongly correlated in eq 2, so that fitting Lapp AB as a function of [Q] cannot give safe values for LAB and Kbind.25 This problem can be, at least in part, overcome by the simultaneous use of all the information contained in the eight isotherms of Table 1. The equilibrium constant of a given process is related to the variation of the thermodynamical parameters in the standard
state according to
( )
K ) exp -
(
)
∆G° ∆S° ∆H° ) exp kBT kB kBT
(5)
where kB is the Boltzmann constant. From eq 5, taking into account the temperature dependence of both LAB and Kbind, eq 2 can be written as
exp app LAB )
(
) (
)
∆SAB° ∆HAB° ∆Sbind° ∆Hbind° exp [Q] kB kBT kB kBT ∆Sbind° ∆Hbind° 1 + exp [Q] kB kBT (6)
(
)
∆H° and ∆S° of both the processes are, in principle, function of the temperature. Previous investigation in aqueous LDAO dispersions have, however, demonstrated that ∆HAB° and ∆SAB° are temperature independent in RCs from Rb. sphaeroides with native ubiquinones.6,26,27 On the basis of this observation and nonsystematic data reported in the literature,28 we assume that the variations of enthalpy and entropy of both processes are constant over the temperature range investigated. The validity of such an assumption will be further discussed below. In order to obtain the values of ∆HAB°, ∆Hbind°, ∆SAB°, ∆Sbind°, and τAP, the τS ≡ τP values of Table 1 as a function of [Q] for the different values of temperature have been simultaneously fitted to eqs 4 and 6. The τP values were weighted for the experimental uncertainty, and in a first step τAP was fixed at 113 ms (average τF coming from the global analysis and from the monoexponential fit of the P+QA- recombination measured in the presence of TBT). Following a first set of iterations that yielded guessed values for ∆HAB°, ∆Hbind°, ∆SAB°, and ∆Sbind°, this constraint was released and the full set of parameters was determined. As shown in Figure 4, the agreement between the experimental data and the fit obtained following this procedure is good. The corresponding values of the parameters are listed in Table 2.
Binding of Ubiquinone to Photosynthetic RCs
J. Phys. Chem. B, Vol. 101, No. 39, 1997 7855
Figure 4. Temperature and quinone concentration dependence of the lifetimes (ms) of charge recombination from P+QB- obtained by global analysis. The surface was calculated according to eqs 4 and 6 using the data reported in Table 2. The points are the experimental τS values of Table 1.
TABLE 2: Lifetime of P+QA- Recombination and Thermodynamical Parameters of the QA- f QB Electron Transfer and Quinone Binding at the QB Site Obtained by a Simultaneous Global Analysis of the Data of Table 1 τAP (ms) ∆H° (eV/molecule)a ∆S° (eV/molecule K)a ∆G° (eV/molecule)a,b log(KD )a,c
binding
electron transfer
-0.526 ( 0.058
112 ( 13 -0.140 ( 0.016
-1.36 × 10-3 ( 2.3 × 10-4 -0.121 ( 0.010
-2.01 × 10-4 ( 6.7 × 10-5 -0.080 ( 0.003
-2.05 ( 0.17
a
Values refer to molar standard state. b ∆G° were calculated at 298 K from the corresponding ∆H° and ∆S° by straightforward calculations. However, ∆H° and ∆S° are very highly correlated in the relationship ∆G°) ∆H° - T∆S°.36 In view of this, the 95% confidence intervals for ∆G°’s were obtained by means of grid search procedures. c KD ) dissociation constant (M) at QB site; T ) 298 K.
As an alternative to the fitting procedure described above, it is possible to fit simultaneously only two isotherms at a time, obtaining a set of parameters that are, of course, characterized by larger uncertainties. It is interesting to apply this procedure to pairs of neighboring isotherms, since, over such a small temperature range (5 °C), all the parameters are likely to be constant. The values of ∆HAB°, ∆Hbind°, ∆SAB°, and ∆Sbind° obtained from pairs of binding curves are plotted in Figure 5 as a function of the average temperature. For both processes (electron transfer and Q binding), the obtained ∆H and ∆S values do not show any temperature dependence, thus confirming the validity of a simultaneous fit of all the isotherms. The global fitting procedure yields a τAP value that is in quite good agreement with the lifetime of the P+ decay measured in the presence of TBT. This finding further supports the presence in reverse micelles of two RC populations, one lacking QB functionality and the other experiencing fast exchange of quinone at the QB site. Furthermore, the numerical value obtained for τAP is in good agreement with the lifetime of P+QArecombination found in aqueous systems,20 suggesting that the
Figure 5. Enthalpy (upper panel) and entropy (lower panel) changes for the QA- f QB electron transfer (closed symbols) and quinone binding to the QB site (open symbols) as a function of the temperature. The thermodynamic parameters were obtained by simultaneous fit according to eqs 4 and 6 of two neighboring isotherms. The corresponding abscissa is the average temperature.
arrangement of cofactors up to QA and the spatial structure of the protein are essentially the same in reverse micelles. The values reported for ∆HAB° in aqueous systems for different buffers and LDAO concentrations indicate a rather large spreading, i.e., -88 meV,6 -150 meV,26 and -220 meV.25 As pointed out by Mc Comb et al.,6 in aqueous systems it can be problematical to unambigously separate the binding and the electron transfer contributions for the highly hydrophobic ubiquinone-10, since the temperature dependence of the micellar properties prevents easy access to thermodynamic parameters. In particular, the mobility of larger quinones (mediated by the micellar dynamics) increases substantially with the temperature so that a transition from the slow-exchange to the fast-exchange case was found.6 Our analysis properly separates the binding and electron transfer parameters, giving safe values of ∆HAB° and ∆SAB° for RCs reconstituted with ubiquinone-10 in reverse micelles. The data of Table 2 are in very good agreement with the values found by Mancino et al.26 (∆HAB° ) -150 ( 11 meV and ∆SAB°) -0.266 ( 0.03 meV). Furthermore, the value of LAB at 25 °C (22 ( 3 obtained from the ∆GAB° of Table 2) is in perfect agreement with a recent re-evaluation of the electron transfer constant in aqueous LDAO systems, performed on the basis of a model that takes into account the nonuniform distribution of quinone molecules in the direct micelles.29 The closeness that exists between ∆HAB°, ∆SAB°, and LAB values in aqueous and in reverse micellar systems supports the
7856 J. Phys. Chem. B, Vol. 101, No. 39, 1997 idea that also the QB site of the RC is not substantially affected in reverse micelles as compared to the water systems. In contrast with the large amount of data concerning the electron transfer to QB, little is known on the temperature dependence of the quinone binding at the QB site. In aqueous systems a rough determination of ∆Hbind°, ∆Sbind° was reported only for ubiquinone-0,6 suffering from an unclear temperature dependence of the partition of Q-0 between aqueous and micellar phase. Studies in reverse micelles lack any temperature dependence of the binding constant, and only relative ∆Gbind° values were determinated for isoprene-substituted quinones.8 The accurate analysis of the temperature and [Q] dependence of the charge recombination kinetics shows that the binding to the QB site is enthalpically driven and substantially opposed by entropic contributions. A similar behavior has been found for the binding of ubiquinones with less than six isoprene units to the QA site of RCs solubilized in aqueous LDAO solutions, while, for long tailed ubiquinones (such as ubiquinone-10) the net binding was found to be driven entirely by a large entropic term.6 From ∆Hbind° and ∆Sbind°, a dissociation constant (KD,QB) for the QB site in n-hexane is obtained equal to (9.0 ( 3.5) × 10-3 M (see Table 2). This value can be profitably compared to that measured in aqueous systems on the basis of the following considerations. At 298 K the ratio (KD,QA/KD,QB)RM between the values found in n-hexane reverse micelles for the dissociation constant at the QA site (2 × 10-4 M)8 and that at the QB site (from Table 2) is close to (KD,QA/KD,QB)LDAO,0.1% (ratio between the constants found in 0.1% LDAO in water6 for the dissociation at QA (0.03 µM) and QB (1.7 µM) sites). The changes in the Gibbs free energy, upon binding of quinone to the same site of RC measured in different media, can be related taking into account the Gibbs free energy of transfer of each chemical species involved in the binding process.7 When we consider the binding in reverse micelles and in LDAO direct micelles we obtain
∆G°bind,RM - ∆G°bind,LDAO ) ∆G°tr,RCQ - ∆G°tr,RC ∆G°tr,Q (7) where ∆G°tr,Q is the standard Gibbs free energy change related to the transfer of ubiquinone from LDAO direct micelles to n-hexane reverse micellar solution. The term [∆G°tr,RCQ ∆G°tr,RC] represents the contribution of ligand binding-induced alterations in the solvation properties of the protein and was found to be null for binding at the QA site;7 thus, eq 7 becomes
∆G°bind,RM - ∆G°bind,LDAO ) RT ln KD,RM RT ln KD,LDAO ) -∆G°tr,Q (8) This relation holds for both QA and QB binding processes as long as ∆G°tr,RCQ - ∆G°tr,RC ) 0 also in the case of binding to the QB site. Under this condition, the ratios between the dissociation constants at QA and QB sites found in n-hexane and LDAO solutions are expected to be the same, as was experimentally found. We conclude therefore that binding of quinone to the QB site does not induce relevant changes in the protein structure. In turn, this allows evaluation of ∆G°tr,Q on the basis of binding at QB. In order to compare values of the dissociation constant in n-hexane and in aqueous LDAO we have used the concentration (M) referred to the hydrophobic phase.30 Accordingly the dissociation constants measured in n-hexane remain unchanged, while the values reported for 0.1% LDAO in water, expressed as overall concentrations, have been divided by a term31 ([LDAO]Vm), where [LDAO] is the molar concentration of detergent (4.36 mM) and Vm is its molar volume
Mallardi et al. (0.2557 L/mol).32 The ∆G°tr,Q estimated in this way includes the hydrophobic effect as well as losses of favorable interactions with the polar heads of LDAO that are absent in the n-hexane phase.7 Using the dissociation constants reported in refs 6 and 8 and the value determined by us for KD,QB in n-hexane, eq 8 yields very close values of ∆G°tr,Q when applied to the QA and the QB binding, i.e. ∆G°tr,Q ) -5.10 kJ/mol and ∆G°tr,Q ) -4.44 kJ/mol respectively. The average value (-4.77 kJ/mol) sounds reasonable.33 Thus, the differences experimentally observed for the binding of quinone to the QB site of RC in n-hexane and aqueous detergent dispersions can be satisfactorily interpreted in terms of transfer free energy of the quinone molecules. This means that the direct quinone interactions within the QB protein binding site, as well as the losses of quinone translational and rotational entropy associated with the binding,34 are essentially the same in n-hexane and in water systems as previously found for the binding to the QA site.7,8 It should be noted that in the investigation performed in the LDAO aqueous systems, all the experimental approaches35 confirmed that the binding equilibrium of ubiquinone-10 at the QB site is slow compared to the charge recombination from P+QA- state. In other words, the dissociation constant measured in LDAO defines the binding to the RC in the PQA state. On the contrary, the quinone in n-hexane being in a “fast-exchange” with the quinone molecule bound at the QB site, the values in Table 2 concern the binding to the RC in the P+QA- state. In view of this, we can conclude that the binding affinity of ubiquinone10 at the QB site of the RC is not markedly different in the dark (PQA) and light (P+QA-) states of the protein. Conclusions In reverse micellar solutions ubiquinone is in fast exchange between the QB site of the bacterial RC and the organic bulk. By monitoring the P+ recovery following a single turnover flash at different temperatures and quinone concentrations, we were able to determine the enthalpy and the entropy changes of both the binding and electron transfer processes. A comparision of the results obtained in reverse micelles of phospholipids in n-hexane and in LDAO (direct) micellar solutions indicates that the quinone affinity for the QB site is approximately the same in the PQA and P+QA- states of the reaction center. The investigation was carried out using ubiquinone-10 in a system where the strong hydrophobic effects are absent and where the influence of the micellar dynamics is negligible. For this reason the information obtained should reflect the thermodynamics governing the Q-exchange in native membrane. Acknowledgment. Dr. A. Hochkoeppler and Dr. F. Francia are kindly acknowledged for their helpful suggestions. One of the authors (G.P.), in particular, specially thanks Dr. S. Sykora for the stimulating discussion about the data analysis. This work was supported by Consiglio Nazionale delle Ricerche (CNR) and by Ministero della Ricerca Scientifica e Tecnologica (MURST). References and Notes (1) Crofts, A.; Wraight, C. A. Biochim. Biophys. Acta 1983, 726, 149. (2) Feher, G.; Allen, J. P.; Okamura M. Y.; Rees, D. C. Nature 1989, 33, 111 and references therein. (3) Mc Pherson, P. H.; Okamura, M. Y.; Feher, G. Biochim. Biophys. Acta 1990, 1016, 289. (4) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. Nature 1985, 318, 618. Michel, H.; Epp, O.; Deisenhofer, J. EMBO J. 1986, 5, 2446. Chang, C. H.; Tiede, D. M.; Tang, J.; Smith, U.; Norris, J.; Schiffer, M. FEBS Lett. 1986, 205, 82. Allen, J. P.; Feher, G.; Yeates, T. O.; Komiya,
Binding of Ubiquinone to Photosynthetic RCs H.; Rees, D. C. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5730. Deisenhofer J.; Michel, H. EMBO J. 1989, 8, 2149. (5) Ermler, U.; Fritzsch, G.; Buchanan, W.; Michel, H. Structure 1994, 2, 925. Fritzsch, G.; Ermler, U.; Michel, H. In Photosynthesis: from Light to Biosphere; Mathis, P., Ed.; Kluwer AP: The Netherlands, 1995; Vol. 1, p 599. (6) Mc Comb, J. C.; Stein, R. R.; Wraight, C. A. Biochim. Biophys. Acta 1990, 1015, 156. (7) Warncke, K.; Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 2920. (8) Warncke, K.; Gunner, M. R.; Braun, B. S.; Gu, L.; Yu, C. A.; Bruce, M.; Dutton, P. L. Biochemistry 1994, 33, 7830. (9) For a review, see: Walde, P.; Giuliani, A. M.; Boicelli, C. A.; Luisi, P. L. Chem. Phys. Lipids 1990, 53, 265. (10) Gray, K. A.; Wachtveil, J.; Breton, J.; Oesterhelt, D. EMBO J. 1990, 9, 2061. (11) Schonfeld, M.; Montal, M.; Feher, G. Biochemistry 1980, 19, 1535. (12) Kendall-Tobias, M. W.; Celis, H.; Almanza, C. S.; Crofts, A. R. Biochim. Biophys. Acta 1981, 635, 585. (13) Agostiano, A.; Catucci, L.; Della Monica, M.; Mallardi, A.; Palazzo, G.; Venturoli, G. Bioelectrochem. Bioenerg. 1995, 38, 25. (14) Nelder, J. A.; Mead, R. Comput. J. 1965, 7, 308. (15) Press, W. H.; Flannery, B. P.; Teulkolsky, S. A.; Vetterling, W. T. Numerical Recipes; Cambridge University Press: Cambridge, 1986 and references therein. (16) Johnson, M. L.; Faunt, L. M. Methods Enzymol. 1992, 210, 1 and references therein. (17) Shinkarev, V. P.; Wraight, C. A. In The Photosynthetic Reaction Center; Deisenhofer, J., Norris, J. R., Eds.; Academic Press: New York, 1993; Vol. 1, p 93. (18) Mallardi, A.; Angelico, R.; Della Monica, M.; Giustini, M.; Palazzo, G.; Venturoli, G. In Photosynthesis: from Light to Biosphere; Mathis, P., Ed.; Kluwer AP: The Netherlands, 1995; Vol. 1, p 843. (19) Kleinfeld, D.; Okamura, M. Y.; Feher, G. Biochim. Biophys. Acta 1984, 766, 126. (20) Ortega, J. M.; Mathis, P.; Williams, J. C.; Allen, J. P. Biochemistry 1996, 35, 3354 (21) See appendix I of ref 17. (22) See, for example: Beechem, J. M. Methods Enzymol. 1992, 210, 37 and references therein.
J. Phys. Chem. B, Vol. 101, No. 39, 1997 7857 (23) An accurate fit of the P+ decay in presence of TBT requires two exponentials with a ratio between the rate constant ranging from 2 to 5, in agreement with the results of Ortega and Mathis in aqueous solution.20 (24) Palazzo, G.; Giustini, M.; Mallardi, A.; Colafemmina, G.; Della Monica, M.; Ceglie, A. Prog. Colloid Polym. Sci. 1996, 102, 19. (25) In the present case the relative uncertainities of LAB and Kbind determined from a two-parameter nonlinear regression of the data corresponding to a single binding isotherm are around 100%. This is the main reason why a preliminary attempt18 to extract ∆HAB°, ∆SAB°, ∆Hbind°, and ∆Sbind° from a sequential analisys of two close isotherms (25 and 31 °C) resulted in incorrect values. (26) Kleinfeld, D.; Okamura, M. Y.; Feher, G. Biophys. J. 1982, 37, 110a. (27) Mancino, L. J.; Dean, D. P.; Blankenship, R. E. Biochim. Biophys. Acta 1984, 764, 46. (28) The constants for the binding of ubiquinone-0 to the QB site of RC solubilized in LDAO and triton water solutions give a satisfactory Van’t Hoff plot, see ref 6. (29) Shinkarev, V. P.; Wraight, C. A. Biophys. J. 1997, 72, 2304. (30) This choice is justified by the following observations: (i) dissociation constants (at the QA site) in n-hexane were found to be independent of reverse micelle concentration; (ii) in aqueous systems hydrophobic quinones behave as expected for complete partition into the detergent phase (for further discussion see ref 6). (31) Strictly, the corrective terms should be expressed in terms of micellar detergent ([LDAO]-cmc)Vm where cmc is the critical micelle concentration. However, in presence of membrane protein the concentration of free detergent molecules is likely to be negligible.6 (32) Milioto, S.; Romancini, D.; De Lisi, R. J. Solution Chem. 1987, 16, 943. (33) To estimate the partitioning of ubiquinone-10 between n-hexane and LDAO, we measured its solubility obtaining 0.44 M in n-hexane and ≈400 µM in 1% LDAO (see Materials and Methods). These values yield ∆G°tr,Q ≈ 6.21 kJ/mol. (34) See, for example: Jenks, W. P. AdV. Enzymol. 1975, 43, 219 and references therein. (35) Namely, amplitude of the fast phase of P+ decay according to eq 1, amplitude of the transient at 397 nm, and assay of RC turnover. For further details, see ref 6. (36) Straume, M. Methods Enzymol. 1994, 240, 89.
