Eur J Biochem 270, 4595–4605 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03845.x Kinetics of the quinone binding reaction at the QB site of reaction centers from the purple bacteria Rhodobacter sphaeroides reconstituted in liposomes Francesco Milano1, Angela Agostiano1,2, Fabio Mavelli2 and Massimo Trotta1 CNR, Istituto per i Processi Chimico-Fisici – Sezione di Bari and 2Dipartimento di Chimica, Universita´ di Bari, Italy Transmembrane proton translocation in the photosynthetic membranes of the purple bacterium Rhodobacter sphaeroides is driven by light and performed by two transmembrane complexes; the photosynthetic reaction center and the ubiquinol–cytochrome c oxidoreductase complex, coupled by two mobile electron carriers; the cytochrome and the quinone This paper focuses on the kinetics and thermodynamics of the interaction between the lipophylic electron carrier ubiquinone-10 and the photosynthetic enzyme reconstituted in liposomes The collected data were simulated with an existing recognized kinetic scheme [Shinkarev, V.P & Wraight, C.A (1993) In The Photosynthetic Reaction Center (Deisenhofer, J & Norris, J.R., eds.), pp 193– The photosynthetic apparatus of the nonsulfur purple bacterium Rhodobacter sphaeroides sits primarily in dedicated portions of the cell membrane called intracytoplasmatic membranes (ICM) [1,2] The key enzymes involved in the build-up of the transmembrane proton gradient [3,4] that eventually trigger ATP synthesis [5] are located in the ICM The increase in the photosynthetic transmembrane proton gradient occurs following absorption of solar electromagnetic radiation, which is performed by light harvesting complexes (LHCs) [6,7] The LHCs channel excitons to the reaction center (RC), a transmembrane enzyme, where they generate a cascade of electron transfer reactions that results in the double reduction of the lipophylic mobile electron carrier, ubiquinone-10 Following reduction the ubiquinone takes up two protons from the cytoplasm, exits the RC and migrates towards the ubiquinol–cytochrome c oxidoreductase (bc1), a second transmembrane complex In the bc1 complex the electrons are utilized to attract two more protons and reduce the cytochrome c2, a water soluble electron carrier that will eventually donate electrons to an Correspondence to M Trotta, Istituto per i Processi Chimico-Fisici – Sezione di Bari, Via Orabona 4-I 70126 BARI, Italy Fax: + 39 080 5442029, Tel.: + 39 080 5442027, E-mail: m.trotta@area.ba.cnr.it Abbreviations: bc1, ubiquinol-cytochrome c oxidoreductase; ICM, intracytoplasmatic membranes; LDAO, lauryl dimethyl amino N-oxide; LHC, light harvesting complex; RC, reaction center Dedication: Dedicated to the memory of Professor Mario Della Monica (Received 18 September 2002, revised 12 September 2003, accepted 22 September 2003) 255 Academic Press, San Diego, CA, USA] and the kinetic constants of the uptake (7.2 · 107 M)1Ỉs)1) and release (40 s)1) processes of the ligand were inferred The results obtained for the quinone release kinetic constant are comparable to the rate of the charge recombination reaction from the state D+QA– Values for the kinetic constants are discussed as part of the overall photocycle, suggesting that its bottleneck may not be the quinone uptake reaction in agreement with a previous report (Gerencser, L., Laczko, G ´ & Maroti, P (1999) Biochemistry 38, 16866–16875) Keywords: reaction center; quinone binding; liposomes; photosynthesis oxidized quinone sitting in the RC, thereby concluding the cyclic electron transport driven by the solar radiation [8] The net result of the entire photocycle is the light-sustained translocation of a proton through the membrane, therefore it is not surprising that a great effort has been made to characterize the mechanism by which the excitons that are absorbed by the RC, excite and shuttle electrons across the enzyme The large amounts of spectroscopic and structural information that have been gathered have enabled a relatively clear description of the electron transfer chain reaction, which is initiated by the absorption of a photon or an exciton The excited electron is transferred from the primary electron donor excited state D* (a dimer of bacteriochlorophyll a), to a chain of electron acceptors located inside the protein at increasing distances from D [9] Due to the spatial organization and the relative energies of the cofactor redox couples, the forward electron transfer reactions occur faster than the recombination reactions and therefore, within hundreds of picoseconds, the electron reaches the primary electron acceptor, ubiquinone-10, sitting in the QA pocket In the absence of exogenous electron – donors (i.e cytochrome) the charge separated state D+QA has a lifetime of 100 ms unless a loosely bound ubiquinone9 10 molecule is present in the QB pocket of the enzyme where – it acts as secondary electron acceptor The state D+QB is more stable, with a lifetime of one or two seconds In the presence of cytochrome, the secondary quinone can allocate a second electron yielded from the absorption of a new photon, thereby functioning as a two-electron gate [3,10] During transfer of the second electron from the primary to the secondary quinone, protons reach the interior of the protein [11] Finally the quinol leaves the RC and is replaced by the oxidized quinone sitting in the membrane pool [12] 4596 F Milano et al (Eur J Biochem 270) Under saturating illumination, the photocycle time scale is in the order of milliseconds A key role in the photocycle is played by the exchange of the two redox forms of the quinone, between the protein interior and the bilayer Some considerations regarding the exchange reaction for the oxidized quinone are made in this paper, based on investigations into the charge recombination reactions that take place in purified RCs reconstituted in proteoliposomes, and in the absence of exogenous electron donors Proteoliposomes were selected because they can be considered a good mimicking system for the photosynthetic membrane, in which the relative amounts of enzyme and quinone can be altered easily, in contrast to the isolated ICM, called chromatophores, where changing quinone concentration is a laborious task [13] Moreover, in the ICM the presence of the entire and active electron transport chain would require the use of decouplers in order to focus the RC–quinone interaction A final consideration for using liposomes is that the solubilizing environment may play a role, particularly when the QB pocket is under investigation [14,15] In this work, RCs were reconstituted in phosphatidylcholine liposomes, which are recognized for producing the best results in the formation of small unilamellar vesicles The kinetics and equilibrium of the exchange between the QB pocket and the quinone pool were estimated The collected data were simulated with the well-known kinetic scheme of Shinkarev & Wraight [16], and the kinetic constants of the ligand uptake (kin) and release (kout) processes were inferred The single species time evolution involved in the kinetic scheme was extracted from the output of the numerical simulation Recombination reactions were also compared to different solubilizing environments such as reverse and direct micelles Materials and methods Isolation of reaction centers and QB site depletion Ó FEBS 2003 taking the baseline recorded before the flash as the starting value Even at high quinone concentrations, the trace deconvolution was obtained with a high correlation coefficient (r2) using bi-exponential functions A drift of less than 1.5% was observed in samples illuminated by the sole measuring beam in the time range of the experiments Each point in the data shown below is the average of three different liposome preparations Reaction center reconstitution in proteoliposomes RC reconstitution in liposomes was accomplished following the procedure outlined in [19–21] One to eight milligrams of 1,2-diacyl-sn-glycero-3-phosphocholine (used at 48% purity, Sigma) were dissolved in 500 lL of chloroform to which, when needed, aliquots of a mM ubiquinone-10 (Sigma) solution were added The resulting solution was carefully dried under a stream of nitrogen in an Eppendorf tube, to form an evenly distributed film of lipids Five hundred microlitres of a 4% (w/v) sodium cholate solution (Sigma) in phosphate buffer, pH 6.8, 100 mM KCl were added to the lipid film Lipids were solubilized by 10–20 repeated one-second sonications (Sonifier Mod 250, Branson Ultrasonic Corporation, Danbury, CT, USA) to form a homogenous solution This solution was added to the QB site-depleted RC (90 lM), shaken vigorously and stored for 15 at °C Finally, the solution was loaded onto a 15 cm Sephadex G-50 Superfine column (Pharmacia) previously equilibrated with the phosphate buffer The band containing RC incorporating liposomes elutes rapidly, and optical measurements were carried out Proteoliposomes were prepared with different quinone/RC (Q/RC) ratios while still maintaining a constant enzyme concentration The RC orientation in the liposome bilayer was inferred from the decrease in the total amount of photobleaching at 865 nm before and after the addition of reduced cytochrome c (Sigma) The two possible orientations of RCs were found to be equally distributed Reaction centers were isolated from Rhodobacter sphaeroDynamic light scattering measurements The hydroides strain R-26.1 following the procedure illustrated by dynamic diameter of liposomes was determined by means of Isaacson et al [17] Protein purity was established using the dynamic light scattering using a Brookhaven Instruments ratio of absorbance at 280 and 802 nm (A280/A802), which Corporation goniometer (BI-200SM) (New York, USA) was kept below 1.3, and the ratio of absorbance at 760 and equipped with a helium/neon laser source (wavelength 865 nm (A760/A865), which was equal to or lower than The 632.8 nm) Samples were contained in cylindrical optical average quinone content was 1.8 when defined by (Q/RC) cells with a diameter of cm while an external thermostat Depletion of the QB site was accomplished using the maintained the temperature at 20.0 ± 0.1 °C All dynamic procedure of Okamura et al [18], with the final preparalight scattering determinations were made at a scattering tions exhibiting a quinone content (Q/RC) ¼ 1.05 ± 0.05 angle of 90° Data were acquired within the 1–104 ns decay as determined by the charge recombination decay No time range that is necessary to determine the signal from changes to the photobleaching amplitude were observed particles upon addition of quinone Charge recombination kinetics were recorded at 865 nm 30 The diffusion coefficient D, was extracted from the measured autocorrelation function by a cumulants method using a kinetic spectrophotometer implemented with an [22,23] using BI-PCSW SIMPLE CUMULANTS software (BrookHamamatsu R928 photomultiplier (Hamamatsu Photonics K.K., Hamamatsu City, Japan), and a Nd-Yag Laser haven Instruments Corporation, New York, USA) (Quanta System, Milan, Italy) which was used for RC In this method, the logarithm of the correlation function, photoexcitation Data were collected onto a Digital 31 g(s), fits to a power series of the correlation time (s): Oscilloscope (Tektronix, Inc., TKS3052, Beaverton, OR, ln fgsịg ẳ A ỵ Bs ỵ Cs2 ỵ ::: USA) and trace deconvolution was performed using software developed in-house The decay traces were where A is a constant that depends on the instrument recorded until complete recovery occurred following setting and photobleaching Absorbance changes were measured Ó FEBS 2003 Quinone exchange in liposomes (Eur J Biochem 270) 4597 39 eluted from the column [19] The RC elutes in a single sharp band that coincides with the lipid elution, indicating that the Q ¼ [4p · n · sen(Q/2)/k], with Q being the modulus of 40 proteins are completely reconstituted into liposomes the scattering vector, n being the refraction index of the solution, k being the wavelength and Q/2 being the Results and discussion 33 scattering angle); and C is equal to 21 Z The kinetic scheme and data analysis 14  ðC À CÞ CðCÞdC5 The reaction scheme outlined in Fig shows the kinetic constants for the final electron acceptor reactions The reactions take place in the neutral state (lower row), and in where, C and C(C) are the decay velocity and the decay the charge separated state that is generated in the RC velocity distribution, respectively) The ratio C/B repfollowing the absorption of a photon in the absence of an resents the size polydispersity distribution exogenous electron donor (upper row) Several descriptions In the hypothesis that particles behave like hard spheres of the scheme are available, the most detailed of which was the average hydrodynamic radius (R) was calculated from given by Shinkarev & Wraight [16] D using the Stokes–Einstein equation, In the dark the RCs undergo a binding equilibrium in R ¼ kB T=6pgD which the loosely bound quinones are taken up and released from the QB site [12] After a short light pulse, the RCs where g is the water viscosity, kB is the Boltzmann undergo a charge separation process, where an electron is constant and T is the absolute temperature transferred from D to a primary quinone acceptor located in The geometry of the liposomes is in agreement with that the QA binding site For proteins in which the QB pocket is obtained by Palazzo et al [24] for liposomes prepared in the empty, a charge recombination occurs with a phenomensame way Combining the parameters obtained for the ological monoexponential decay constant [9] k ¼ kAD preparation of liposomes as summarized in Table 1, it is 41 which is % s)1 (k is the phenomenological delay F constant F possible to estimate a RC/liposome ratio of 500 ± 150 of the fast phase and kAD is the back electron transfer depending on the lipid/protein ratio used to prepare the constant from the D+QA– and D+QA–QB states) In RCs liposomes (see below) These values correspond to an RC which have the QB pocket occupied, the electron rapidly surface concentration ranging from 2.7 to 20.0 nmolỈm2 equilibrates between the two final acceptors with an equilibThe lower concentration is in agreement with 3.0 nmolỈm2 42 rium constant (L ) that can be expressed as L ¼ AB AB calculated for chromatophores assigned a radius of 50 nm kAB/kBA (kAB being the forward electron transfer constant [25,26] and using the 50–60 RC/chromatophore ratio as from D+QA–QB to D+QAQB– and kBA being the backward found by Saphon et al [27] electron transfer from D+QAQB– to D+QA)QB) When the It is well known that the radius of liposomes is influenced QB pockets are fully occupied, the charge recombination 36 by the molar ratio of lipid/detergent in the mixed micelles reaction is also monoexponential, with a phenomenological starting solution, and in our preparations this ratio was rate constant, ks: always below the critical value of 1.33 at which the transition between the extended bilayer sheet and the micelle takes place Each of the above described experiments exhibits no significant variation in the diameter of the liposomes with varying lipid/detergent molar ratio Due to dispersion of the data for the same sample we conclude that an average value of 110 ± 25 nm can be 37 assumed as a reasonable estimate of the liposomes radius The measurements made on both liposomes containing the RC (proteoliposomes), and pure liposomes (not containing protein), gave substantially the same results Reconstitution of the protein was confirmed by preparing liposomes in the presence of a fluorescent lipid (1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino]Fig The kinetic scheme for reaction centers in the presence of quinone 38 dodecanoyl]-sn-glycero-3-phosphocholine purchased from association and dissociation (quinone exchange), both in the dark and in Avanti Polar Lipids Inc., Alabaster, AL, USA), and the charge separated state The constants in the scheme are defined as recording the visible spectra and fluorescence of the solution + –  B ¼ ÀC ¼ DQ2 Table Parameters of proteliposomes preparations RC area assumes a horizontal section as an ellipse [9] of 0.3 · 0.4 nm2 Liposome radius derived from experimental data RC area (nm2) Liposome radius (nm) Liposome area (nm2) (Liposome area/RC area) 10 110 ± 25 (1.5 ± 0.6) · 105 1.5 · 104 follows: kAD ¼ back electron transfer constant from the D QA and – D+QAQB states, assuming that the charge recombination process from QA– is not affected by the functional occupancy of the QB site; kin ¼ quinone uptake constant; kout ¼ quinone release kinetic constant; kAB ¼ forward electron transfer constant from D+QA–QB to D+QAQB–; kBA ¼ backward electron transfer from D+QAQB– to D+QA–QB The direct recombination route from D+QAQB– is not shown as its constant is negligible compared to the others kin and kout are assumed to be independent of the redox state of QA (see text for discussion) Ó FEBS 2003 4598 F Milano et al (Eur J Biochem 270) Fig Fraction of slow phase obtained by fitting Eqn (1) to the experimental traces d, phosphatidylcholine proteoliposomes prepared with lipid/protein molar ratio of 1000 : 1; [Q]/[RC], concentration of the species in the mixed micelles, where [RC] ¼ 8.3 lM; s, 2.1 lM RC made up in 0.025% LDAO in 20 mM tris buffer pH 8, where the 64 quinone is solubilized in Triton X-100  ðkAD þ kBD LAB Þ 1 þ LAB   % kAD 1 ỵ LAB  Eqn 1ị This approximation holds because the direct recombination reaction from the D+QAQB– state has a negligible kinetic constant (kBD < 0.1 s)1) [28,29] In the presence of a subsaturating quinone concentration, only a fraction of the QB sites can be filled and the decay can be fitted with the sum of two exponential decays: DAtị ẳ DA0 expkF tị ỵ DA0 expkS tÞ Eqn ð2Þ fast slow where t is the time, DA(t) represents the amplitude at any instant t, and DAfast and DA0 represent the amplitudes of slow the fast and slow phase respectively Proteoliposomes were prepared using QB depleted reaction centers in the presence of increasing amounts of 45 ubiquinone-10, the naturally occurring quinone in the QB site Charge recombination kinetics were recorded and time evolution traces of absorbance changes were fitted (r2 > 0.995) using Eqn (2) where kF and kS represent the phenomenological decay constants of the fast and slow phase, respectively In this work the kF constant is assumed equivalent to the kinetic constant kAD (8.3 s)1) of the decay from the QA– containing states (Fig 1) Indeed, upon addition of inhibitors of QB functionality, the decay of the charge separated state is monoexponential, with a constant slightly faster than the kF (%10 s)1), indicating that the secondary quinone is displaced from its binding site as 46 observed in detergent In contrast, kS results from more than one clear-cut process as discussed below As the quinone/RC ratio 47;48 increases, a rise in the slow phase amplitude and a decrease 47 in the decay constant are observed Figure shows the dependence of the slow phase relative amplitude (titration curve) on the increase of Q/RC Similarly the dependence of the decay constant is shown in Fig Under these conditions the binding reaction has a role in the slow component of the charge recombination The slow Fig Slow phase decay constant as a function of quinone/RC molar ratio d, liposomes; s, detergent decay constant depends both on the rate ratio between the quinone exchange and the charge recombination from the states D+QA– or D+QA–QB, in addition to Q/RC The quinone release rate kout[D+QA)QB] can be normalized to the back electron transfer rate from the appropriate state; kout[D+QA)QB]/kAD[D+QA)QB], and the ratio can be used to describe the quinone exchange regime For instance, if (kout/kAD) > the exchange is defined as fast, whereas for (kout/kAD) < the exchange is defined as slow The different kinetic behaviour of the protein when solubilized in different environments (e.g direct micelles, reverse micelles, and proteoliposomes) comes from the influence played by the surroundings on kin, kout and LAB For instance, in direct lauryl dimethyl amino N-oxide 49 (LDAO) micelles a decay sum of two exponential is observed [18] with a subsaturating quinone concentration [i.e a fast phase with a decay constant (kF ¼ kAD) and a slow phase with a decay constant (kS) given by Eqn (1)], that can be explained only by considering a slow exchange The quinone uptake and release can be neglected during the charge recombination reaction, hence the relative amplitude of the slow phase is proportional to the QB site occupancy On the other hand, in direct Triton X-100 micelles a decay sum of two exponentials is observed [30] with a subsaturating quinone concentration in which the kS depends on the concentration of added quinone, ranging from 1.1 s)1 to 2.7 s)1, showing a fast exchange at the QB site Agostiano et al [31] found that the charge separated state of RCs solubilized in phospholipid reverse micelles will decay as the sum of two exponentials The reverse micelles are dissolved in hexane where the unbound quinone is highly soluble The decay has a kF ¼ kAD and a slow phase with a constant kS decreasing from s)1 to s)1, and a relative amplitude increasing to 1.0 for 400 £ Q/RC £ 7000 Such behaviour was explained in terms of fast quinone exchange Assuming quinone molecules uniformly distributed among vesicles of different sizes, Palazzo et al [24] studied the influence of the spread of the local solute concentration on the phenomenological kinetic constants In the present work the Q/RC ratio ranged from 0.02 to 4, and full QB reconstitution was obtained for values higher than The long chain exogenous quinone was confined to Ó FEBS 2003 Quinone exchange in liposomes (Eur J Biochem 270) 4599 Table Constants and exchange domains for three different solubilizing environments RC solubilizing environment kAD (s)1) LAB KB (M)1) LDAO direct micelles LDAO direct micelles [51] Triton X-100 direct micelles [30] Phospholipids reverse micelles Phosphatidylcholine proteliposomesb 8.3 8.3 8.3 8.3 8.3 9–10a 20 % 6–8a 11.5 15 107 107 % · 107 1.2 · 104 1.8 · 106 65 a Calculated from the equation (kAD/kS) ) using the values kAD ¼ 8.3 s)1 and ks ¼ 0.8 s)1 [16] the liposome bilayers Additionally, as a direct consequence of our liposome preparation method, a solute molecule distribution weighted by the bilayer vesicle volume was considered (i.e larger vesicles will contain larger numbers of solute molecules) As shown in the Appendix, under this assumption the average local volume concentration of quinones is the same for aggregates of all sizes and the polydispersity can be neglected at high overall quinone concentration [Q] In the investigated [Q] concentration range this condition is not fulfilled for the first two values, where the decay from the D+QA– state is predominant Analogously to the previous case, the decay of the charge separated state is fitted by the sum of two exponentials with kF ¼ kAD and a slow phase kS decreasing from 1.5 s)1 to 0.5 s)1 Using the asymptotic kS value in the equilibrium constant, LAB is found to be 15.6 It should be noted that when the condition (kout/kAD) > occurs, the quinone uptake and release take place during the charge recombination reaction The exchange regime and the value of some constants for the three solubilizing environments are summarized in Table Numerical simulations The set of differential equations [Eqn (3)] required for the kinetic scheme shown in Fig was numerically solved by a fourth order Runge–Kutta method Using this approach a value for the quinone uptake and release kinetic constants and therefore the quinone binding constant (KB ¼ kin/kout) can be determined The symbols used in Eqn (3) are the same as those used in [16] Numerical simulations have been carried out for the lipid/protein molar ratio 1000 : by using the values listed in Table Table Numerical value for the constants employed in the simulation of D+ decay LAB is taken from Table 2; kAB and kBD from [28] [29]; kBA is obtained from the assumption that the forward electron transfer constant in proteoliposomes remains unaltered Recently, Taly et al [52] measured, with 10% uncertainty, kAB ¼ 8700 s)1 for the wild type (Rb sphaeroides 2.4.1) in dimyristoylphosphatidylcholine liposomes The numerical simulation has also been tested for different kAB values and it was found to be insensitive for rates in the range 5000 s)1 ) 15000 s)1 Constant kAB kBD kBA 66 kAD b kout/kAD 1 4.8 This work > dx/dt ẳ kAD ỵ kin qịx ỵ kout y > > > > dy/dt ẳ k qx ỵ k z k ỵ k ỵ k ịy > in BA AD AB out > > > < dw/dt ¼ k u þ k x À k qw out AD in Eqn 3ị > > dz/dt ẳ kAB y kBA ỵ kBD ịz > > > du/dt ẳ k qw ỵ k y ỵ k z k u > > in AD BD out > > : dq/dt ¼ Àk qqw ỵ xị ỵ k y ỵ uịq in ỵ in Q =ẵRC; A where x ẳ ẵD y ẳ ẵDỵ Q QB =ẵRC; z ẳ A ỵ ẵD QA QB =ẵRC; w ẳ ẵDQA =ẵRC; u ẳ ẵDQA QB =ẵRC; q ẳ ẵQfree =ẵQtotal and q ẳẵRC=ẵQtotal Immediately after the flash, at time zero, the electron is found only in the charge separated states involving the primary electron acceptor, i.e D+QA and D+QA–QB, while QB has not yet been reached The D+QA–QB state rapidly disappears, with constant kAB generating the state – D+QAQB, until the equilibrium is attained within few milliseconds Simultaneously, the charge separated states start to decay and the different contributions cannot be resolved by monitoring the D+ decay The free quinone concentration ([Q]free) drops from its equilibrium ÔdarkÕ value and is driven to the QB site by the presence of the electron A typical time-evolution obtained by solving Eqn (3) is shown in Fig The quinone binding constant KB was varied in the range · 105 ) · 107 M)1 and the quinone release constant kout was varied between 0.25 and 2500 s)1, spanning from a slow to a fast exchange regime; this is shown in Fig where the charged species decay is simulated for seven kout values at constant KB The slow decay constant ks is weakly dependent on kout for large and small kout/kAD values, whereas the dependence increases when this ratio is close to The overall dependencies of simulated ks and DA0slow upon KB and kout/kAD are illustrated in Fig The kin and kout values that minimize the square-root difference between the simulated and experimental traces were obtained by using the Ôsimple search methodÕ [32] with a tolerance of 10)4 giving kin ¼ 7.2 · 107 M)1ặs)1 and kout ẳ 40 s)1 From the best fit values, KB ¼ 1.8 · 106 M)1 and kout/kAD ¼ 4.8 were obtained The agreement between the experimental and the simulated data for the reconstitution of QB site experiments in proteoliposomes (Fig 7) is very satisfying Value 104 s)1 · 10)2 s)1 6.6 · 102 s)1 8.3 s)1 Discussion An important issue arising from the above experimental and simulated data is the different behaviour of the quinone exchange when passing from direct micelles to 4600 F Milano et al (Eur J Biochem 270) Ó FEBS 2003 Fig Numerical simulation of time evolution following light pulses of D+QA–, D+QA–QB, D+QAQB– and D+Qfree Q/RC ¼ 0.74; [RC] ¼ 8.3 lM; KB ¼ 106 M)1; kout ¼ 25 The initial ten milliseconds of the time-course are shown in the insert Fig Simulated decay of D+ obtained for Q/RC = 0.37 and a binding constant of KB = 106 M)1 Different decays were obtained with different kout The noisy line represents the recorded trace in the experimental conditions used for the simulation proteoliposomes The main difference between these two solubilizing environments is their organization with the enzyme RC–LDAO complexes have been characterized by small angle neutron scattering [33,34] The complex is formed by a toroidally shaped group of micelles surrounding the most hydrophobic part of the protein In these complexes the detergent around the protein is organized with the chain perpendicular to the protein surface and with the terminal region sticking into the protein This reduces the hydrophobic portion of the detergent in which free ˚ quinone can diffuse (% 1500 A3) [33] Crystallographic data [14] shows that the detergent itself is located in the channel into which the quinone isoprenoid chain sits in the enzyme This explains the slow exchange process of the quinone at its binding site Conversely, the dimensions of Triton X-100 micelles [35] are larger than those formed by LDAO, thereby allowing a larger quinone pool size as well as higher ligand mobility The proteoliposomes are topologically similar to the 50 detergent–RC complexes, i.e they are disconnected solubilizing environments, but they differ because proteoliposomes can allocate a large number of proteins, in the order of hundreds of RCs per vesicle As a consequence, the number of quinones per liposome ranges from tens to hundreds, and fluctuations in the local concentration can be neglected Liposomes can therefore be used for drawing general conclusions on quinone binding at the QB site The lipophylic environment represented by proteoliposomes has several advantages in describing the exchange of quinone in photosynthetic membranes compared to the RC–detergent complexes: (a) the quinone is arranged in the bilayer in a similar manner to chromatophores, where quinone can freely diffuse towards and away from the enzyme, and the large volume of the bilayer allows the accommodation a large number of ligands; (b) the arrangement of the lipid molecules around the RC is not known, but it can be reliably assumed that they will not attach with their chains into the protein No direct interaction with the QB site is expected and the channel will always be accessible for the quinone exchange; (c) The absolute value of ks measured in detergent is larger than the one obtained in saturating conditions in liposomes, indicating a relative stabilization of Q À This difference in the semiquinone stability might be B associated with small detrimental changes in the QB pocket, induced by the detergent hydrophobic chains that are absent in the case of liposomes The absence of detrimental effects in liposomes is also conrmed by the D ỵ electron nuclear double resonance spectra as recently reported by our group [19] Some considerations on the absolute value of the quinone exchange constants in proteoliposomes can be useful in order to understand the same process in photosynthetic membranes For a bimolecular reaction of an enzyme with a small ligand, a reasonable approximation of the frequency Ó FEBS 2003 Quinone exchange in liposomes (Eur J Biochem 270) 4601 Fig Three dimensional representation of (A) ks and (B) DA0 dependence on KB and kout/kAD For kout/kAD < (slow exchange), the fraction of slow slow phase coincides with the fraction of occupied QB sites in the dark adapted state The ks obtained under such conditions is independent, as expected, of the concentration of quinone in the solubilizing environment, matching the value from Eqn (1) For kout/kAD > (fast exchange), the fraction of slow phase does not coincide with, and moreover, over-estimates the fraction of QB sites occupied in the dark adapted state fC ẳ 4pr0 DRC ỵ DQ ị 10À3 NA % 4pr0 DQ Á 10À3 NA Eqn ð4Þ Fig Comparison between simulated (s) and experimental values (d) for ks (A) and DA0 (B) as functions of Q/RC slow of collision (fC) in the diffusion controlled regime can be obtained by simple considerations on the mobility of the two species [36]: r0 is the minimum approaching distance in cm, assumed to be equal to the radius of the protein; NA is the Avogadro Number; DRC and DQ represent the diffusion coefficients of the RC and quinone, respectively The approximation in Eqn (4) is based on the large difference in the dimension of the colliding molecules For mitochondrial cytochrome bc1, a diffusion constant D ẳ 4.0 à 10)11 cm2ặs)1 was measured [37,38] and a similar order of magnitude can be expected for the RC, as both are large membrane proteins Several techniques have been used to measure the ubiquinone-10 diffusion coefficient DQ Using fluorescent quenching [39–42] the diffusion coefficient was found to span the range · 10)7 ) · 10)6 cm2Ỉs)1 With the fluorescent recovery after photobleaching technique, a value in the range · 10)8 ) · 10)8 cm2Ỉs)1 was obtained [37,43,44] Electrochemical methods were also used, and a value of (2.0 ± 0.4) · 10)8 cm2Ỉs)1 was obtained [45] According to Blackwell and coworkers [41,42] the DQ obtained using the fluorescence quenching method can be disregarded because it overestimates the actual value Therefore, using DQ ẳ 2.0 à 10)8 cm2ặs)1 in Eqn (4), an fC value equal to 5.0 · 107 M)1Ỉs)1 is obtained It should be noted that although the collision frequency is slightly overestimated because Eqn (4) is valid for three-dimensional systems, this value remains within the accuracy of these considerations Assuming the surface of the QB channel entrance to be the area of the protein where a successful collision can take place [46], an estimate of the association constant can be ˚ ˚ made Assuming % 30 A2 and 5000 A2 for the quinone moiety, and L and M subunit surfaces in contact with the lipids respectively, a correction factor of 0.006 is obtained As a consequence kin can be estimated to be · 105 M)1Ỉs)1 which is very close to the result of the best-fit procedure (kin ¼ 7.2 · 107), corrected by the factor [L]v¢tail (see Appendix) giving kin[L]vÂtail ẳ 3.6 à 105 M)1ặs)1 This suggests that the rate limiting step for the association of the RC 4602 F Milano et al (Eur J Biochem 270) Ó FEBS 2003 limit on the release rate of quinol, which will lead to a 52 and quinone is the diffusion of the latter through the proteoliposomes, implying that the ligand in the binding 53 result larger than the same rate for the oxidized form: (kout)QH2 ‡ (kout)Q Conversely, from the hypothesis that channel, either taken up or released, moves at least as fast as in the bilayer; this can be expressed as (DQ)channel ‡ DQ cytochrome turnover would be unchanged in both the vesicle and in detergent, i.e that the unbinding of oxidized Assuming a random transfer for quinone, it will cover the ˚  cytochrome would remain the slowest step of the photobinding channel of length X (% 50 A) in an average time of  cycle, the upper limit for the quinol release can be set to (1/kdiff) £ (X 2/2DQ) % · 10)5 s (kout)QH2 £ 1000 s)1 The average time for quinone release, obtained from the simulations of 1/kout ¼ 25 ms, accounts for both the residence time in the channel (1/kdiff) and for the time Conclusions required to unbind from the pocket (1/kP): By studying the charge recombination kinetics of reaction 1 1 centers incorporated into liposomes, thermodynamic and ẳ ỵ % ẳ 25 ms Eqn ð5Þ kout kdiff kP kP kinetic parameters have been inferred which regulate the photosynthetic turnover of this important protein These As a consequence, the bottleneck in the quinone release values, although obtained in a simpler environment, can be process is represented by the unbinding of the ligand from reasonably taken as a fair approximation to the ones its pocket The value of 25 ms obtained from Eqn (5), is actually working in the natural ICMs comparable with that of % ms obtained by NMR The high value found for the quinone equilibrium binding measurements for systems kept in the dark in the presence constant KB ¼ (kin/kout) ¼ 1.8 · 106 M)1, makes it possible of ubiquinone-10 [47] The results differ by one order of for the reaction centers to efficiently work with a small magnitude, and the discrepancy can be attributed to the quinone pool: we found that with a quinone/protein molar assumption that the charge separated and neutral RCs ratio as small as three, the QB site was fully occupied When exchange quinones with the same kinetics, regardless of the the electron reaches the QA site in a reaction center without redox state of QA (Fig 1), as we assume that the presence of the quinone in the QB pocket, only the charge recombinathe hydrophobic tail has no influence on kP This suggests tion reaction can occur, which results in a loss of excitation that in the charge separated state the quinone release is energy However, in physiological conditions, where the slower than in the dark This can be explained by invoking quinone pool size has been estimated to be 10 or larger, this the gated propeller twist imposed on QB by the presence of a is very unlikely to happen It would be interesting to negative charge on QA [48] that buries the quinone head in investigate similar reconstituted systems prepared with the inner part of the QB pocket, thereby increasing the RC mutants with smaller charge recombination rate interaction energy between the ligand and the binding site (kAD) constants which fulfil the slow exchange regime in In a forthcoming work the exchange kinetic dependence on 54 liposomes the QA redox state will be addressed The results obtained in this paper can be related to the RC photocycle, when the photochemistry takes place in the Acknowledgements presence of an exogenous electron donor able to doubly The authors are grateful to Professor E Caponnetti and Dr Lucia reduce D+ Gerencser et al [49] have measured the steady´ Pedone of Dipartimento di Chimica Fisica – Universita di Palermo for state rate of cytochrome c turnover in detergent, demonperforming the dynamic light scattering measurements Thanks also to strating that at low ionic strength the reaction of ´ ´ ´ ´ Laszlo Nagy and Peter Maroti for helpful discussions This work was cytochrome c3+ unbinding from the RC is the rate limiting made possible thanks to the financial support of the Grants: )1 )1 step of the photocycle (1000 s < koff < 2000 s ) By Meccanismi Molecolari della Fotosintesi (FIRB-MIUR) and Cofin – employing the simulated kin ẳ 7.2 à 107 M)1ặs)1 value, it is MIUR 2002 possible to estimate the [Q]min at which quinone uptake is not the rate limiting step: kin · [Q]min > 1000 s)1 Þ References [Q]min > 14 lm, which agrees with the value of 25 lM used in Gerencser’s work Such [Q]min can easily be obtained Collins, M.L.P & Remsen, C.C (1991) The purple phototrophic in our preparation and would give a quinone pool of bacteria In Structure of Phototrophic Prokaryotes (Stolz, J.F., Q/RC % 3, which is smaller than the average dimension of ed.), pp 49–77 CRC Press, Boca Raton, FL, USA Oelze, J & Drews, G (1972) Membranes of photosynthetic bacthe quinone pool in chromatophores [50] teria Biochim Biophys Acta 265, 209–239 Interestingly the structure of the QB pocket and the ´ Vermeglio, A., Joliot, P & Joliot, A (1995) Organization of quinone in the illuminated crystals [48] shows a strong electron transfer components and supercomplexes In Anoxygenic interaction between the protein residues and the quinoid Photosynthetic Bacteria (Blankenship, R.E., Madigan, T.M & moiety of the ligand, based on the formation of hydrogen Bauer, C.E., eds), pp 279–295 Kluwer Academic Publisher, bonds These bonds will, of course, disappear following 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Biochemistry 38, 16866–16875 50 Hauska, G & Hurt, E (1982) Pool function behaviour and mobility of isoprenoid quinones In Function of Quinones in Energy Conserving Systems (Trumpower, B.L., ed), pp 87–110 Academic Press, New York, NY, USA 51 Shinkarev, V.P & Wraight, C.A (1997) The interaction of quinone and detergent with reaction centres of purple bacteria I Slow quinone exchange between reaction centre micelles and pure detergent micelles Biophys J 72, 2304–2319 52 Taly, A., Baciou, L & Sebban, P (2002) The DMPC lipid phase transition influences differently the first and the second electron transfer reactions in bacterial reaction centers FEBS Lett 532, 91–96 Appendix In this section the distribution of a highly hydrophobic solute among vesicles of different sizes will be addressed, assuming vesicles to be spherical compartments with bi-layered boundaries of negligible thickness Moreover, the existence of a density probability function P(R) should also be defined as follows: P(R) dR equals the probability to find a VR vesicle with radius between R and R + dR At this level of approximation, the overall concentration of vesicles [V] can be calculated in terms of the lipid concentration [L] by the surface area conservation law per unit volume: Z ẵLa ẳ ẵV 8pR2 PRịdR ẳ ẵV8phR2 i Eqn 6ị a being the lipid head area Additionally, the bilayer volume can be also estimated according to Palazzo et al [24], as the product of the lipid number on the vesicle bilayer surface (8pR2/a) multiplied by the lipid tail volume vtail: Eqn 7ị mRị ẳ 8pR2 =aị mtail The distribution of solute molecules S, among spherical vesicles V, of different radius R, can be then described with the following density function: Pn; Rị ẳ Pn j RịPRị Eqn 8ị where P(n, R) dR is the probability to find n solute molecules inside a VR vesicle (i.e an aggregate of size between R and R + dR), and it equals the products of the probability P(R) dR to find a VR vesicle multiplied by the conditional probability P(n|R) to find n solute molecules in this aggregate As a consequence of Eqn (8) the average number of solute molecules ỈNỉ among vesicles of any size is obtained by the summation over all possible solute molecule numbers and the integration over all vesicle size ranges: Z X Z hNi ẳ nPn j RịPRịdR ¼ hNðRÞiPðRÞdR n Eqn ð9Þ As shown by the previous equation, ỈNỉ can also be expressed in terms of the average numbers of solute molecules among VR compartments: ặN(R)ổ ẳ SnnP(n|R) The term ỈN(R)ỉ can also estimate, in terms of macroscopic concentration, the ratio between the bulk concentration of S molecules contained in VR vesicles ([SR]), divided by the bulk concentration of these aggregates [VR]: hNRịi ẳ ẵSR ẵVR Š Eqn ð10Þ [VR] is directly linked to the overall vesicle bulk concentration [V] by the equation [VR] ¼ [V]P(R) dR, whereas different hypotheses can be found on the relationship between [SR] and [S], depending on the experimental preparation method Herein two main assumptions will be considered: (a) the solute molecule distribution is independent of the vesicle radius, (b) the Ó FEBS 2003 solute molecule distribution is weighted on the volume of the vesicle bilayer v(R) If a random distribution is assumed among vesicles, i.e no dependence on the size is considered, then [SR] ¼ [S]P(R)dR so that by using Eqns (10 and 6) one obtains:      ẵS ẵS 8phR2 i hNRịi ẳ ẳ Eqn 11ị ½VŠ ½LŠ a therefore ỈN(R)ỉ is independent of the specific vesicle radius R It will however, depend on the second moment of the P(R) probability density function: ặR2ổ ẳ ặRổ2 + r2 In fact, at fixed [L] if the average size of vesicles increases then their overall concentration [V] must decrease and ỈN(R)ỉ must increase On the other hand, in the case of the bilayer volume weighted distribution, the concentration [SR] will result:     mðRÞPðRÞdR R ẳ ẵS PRịdRị ẵSR ẳ ẵS R mRịPRịdR hR2 i and by means of Eqns (10 and 6) the average number of solute molecules in R sized vesicles will be:    ẵS 8pR2 hNRịi ẳ Eqn 12ị ½LŠ a in this case ỈN(R)ỉ is proportional to the bilayer vesicle volume Defining the solute vesicle volume concentration as the mole number of solute molecules in a vesicle divided by the bilayer volume: (SR)V ¼ n/(NAv(R)), we can now calculate the average ặ(S)Vổ:  Z X n hSịV i ¼ ðPðn jRÞPðRÞdRÞ NA vðRÞ n Z  hNðRÞi ¼ ðPðRÞdRÞ NA vðRÞ NA being the Avogadro number In scenario (a), by using Eqns (11, and 7) one obtains:   Z  ẵS PRịdR hR2 iị hSịV i ẳ ẵL v0tail R2 !   ẵS hR2 i Eqn 13ị % ẵL vtail hRi2 where vÂtail ẳ vtailNA, as obtained by Palazzo et al [24] On the other hand, in scenario (b) ỈN(R)ỉ can be calculated using Eqns (12, and 7): !      ẵS R2 ẵS 8pR2 ẳ Eqn 14ị hNRịi ẳ ẵV ẵL a hRi2 Quinone exchange in liposomes (Eur J Biochem 270) 4605 and the average solute concentration will result:    ẵS hSịVi ẳ Eqn 15ị ẵL mtail The previous equation clearly shows that in the case of a solute distribution weighted on the bilayer volume, the average vesicle volume concentration of solute Ỉ(S)Vỉ is independent of the vesicle size, and the experimental results from different vesicle size distributions can be directly compared Moreover, at fixed lipid concentration [L], Ỉ(S)Vỉ is proportional to the bulk solute concentration [S] and this allows one to use this value in the kinetic equations, keeping in mind that the bimolecular kinetic constants used in Eqn (8) must be corrected multiplied by [L]v¢tail to obtain the real constants Another important point is to estimate the standard deviation of Ỉ(S)Vỉ and this can be done by first calculating: 2 ! Z X n hSịV i ẳ NA mRị n PnjRịPRịdRị ẳ hSịV i2 ị     ẵS a ỵ ẵL 8pm02 R2 tail and then obtaining the polydispersity index:       hS i ẵL a 1ẳ PhSi ẳ ẵS 8p R2 hSi2     ½LŠ a % ½SŠ 8p hR2 i whilst keeping in mind Eqn (15) PỈSỉ shows that by increasing the bulk solute concentration or the average radius of liposomes, the (S) polydispersity decreases In the studied case the RC concentration gives: Ỉ(RC)Vỉ molỈL)1 [RC] 67 molỈL)1 )6 8.3 · 10 rỈ(RC)ỉ molỈL)1 )3 1.5 · 10 7.1 · 10 PỈ(RC)ỉ )5 2.2 · 10)3 whereas for the quinone we obtain: Ỉ(Q)Vỉ molỈL)1 [Q]total molỈL)1 )7 1.5 · 10 3.1 · 10)5 rỈ(Q)ỉ molỈL)1 )5 2.7 · 10 5.6 · 10)3 PỈ(Q)ỉ )6 9.5 · 10 1.3 · 10)4 1.2 · 10)1 5.9 · 10)4 showing that only at very low concentration of quinones, the spread of the concentration distribution becomes not negligible  ... data [14] shows that the detergent itself is located in the channel into which the quinone isoprenoid chain sits in the enzyme This explains the slow exchange process of the quinone at its binding. .. regardless of the the electron reaches the QA site in a reaction center without redox state of QA (Fig 1), as we assume that the presence of the quinone in the QB pocket, only the charge recombinathe... primary quinone acceptor located in The geometry of the liposomes is in agreement with that the QA binding site For proteins in which the QB pocket is obtained by Palazzo et al [24] for liposomes