Kinetics of the quinone binding reaction at the QB site of reactioncenters from the purple bacteria Rhodobacter sphaeroidesreconstituted in liposomes

Francesco Milano1, Angela Agostiano1,2, Fabio Mavelli2 and Massimo Trotta1

1CNR, Istituto per i Processi Chimico-Fisici – Sezione di Bari and 2Dipartimento di Chimica, Universita di Bari, Italy

Transmembrane proton translocation in the photosyntheticmembranes of the purple bacterium Rhodobacter sphaero-ides is driven by light and performed by two transmem-brane complexes; the photosynthetic reaction center and theubiquinol–cytochrome c oxidoreductase complex, coupledby two mobile electron carriers; the cytochrome and thequinone. This paper focuses on the kinetics and thermo-dynamics of the interaction between the lipophylic electroncarrier ubiquinone-10 and the photosynthetic enzymereconstituted in liposomes. The collected data were simula-ted with an existing recognized kinetic scheme [Shinkarev,V.P. & Wraight, C.A. (1993) In The Photosynthetic Reac-tion Center (Deisenhofer, J. & Norris, J.R., eds.), pp. 193–

255. Academic Press, San Diego, CA, USA] and the kineticconstants of the uptake (7.2 · 107 M

)1Æs)1) and release(40 s)1) processes of the ligand were inferred. The resultsobtained for the quinone release kinetic constant are com-parable to the rate of the charge recombination reactionfrom the state D+QA

–. Values for the kinetic constants arediscussed as part of the overall photocycle, suggesting that itsbottleneck may not be the quinone uptake reaction inagreement with a previous report (Gerencser, L., Laczko,G.& Maroti, P. (1999) Biochemistry 38, 16866–16875).

Keywords: reaction center; quinone binding; liposomes;photosynthesis.

4The photosynthetic apparatus of the nonsulfur purplebacterium Rhodobacter sphaeroides sits primarily in dedica-ted portions of the cell membrane called intracytoplasmaticmembranes (ICM) [1,2]. The key enzymes involved in thebuild-up of the transmembrane proton gradient [3,4] thateventually trigger ATP synthesis [5] are located in the ICM.The increase in the photosynthetic transmembrane protongradient occurs following absorption of solar electromag-netic radiation, which is performed by light harvestingcomplexes (LHCs) [6,7]. The LHCs channel excitons to thereaction center (RC), a transmembrane enzyme, where theygenerate a cascade of electron transfer reactions that resultsin the double reduction5 of the lipophylic mobile electroncarrier, ubiquinone-10. Following reduction the ubiquinonetakes up two protons from the cytoplasm, exits the RC andmigrates towards the ubiquinol–cytochrome c oxidoreduc-tase (bc1), a second transmembrane complex. In the bc1complex the electrons are utilized to attract two moreprotons and reduce the cytochrome c2, a water solubleelectron carrier that will eventually donate electrons to an

oxidized quinone sitting in the RC, thereby concluding thecyclic electron transport driven by the solar radiation [8].The net result of the entire photocycle is the light-sustainedtranslocation of a proton through the membrane, thereforeit is not surprising that a great effort has been made tocharacterize the mechanism by which the excitons that areabsorbed by the RC, excite and shuttle electrons across theenzyme. The large amounts of spectroscopic and structuralinformation that have been gathered have enabled arelatively clear description of the electron transfer chainreaction, which is initiated by the absorption of a photon oran exciton. The excited electron is transferred from theprimary electron donor excited state D* (a dimer ofbacteriochlorophyll a)6 , to a chain of electron acceptorslocated inside the protein at increasing distances fromD [9].Due to the spatial organization and the relative energies ofthe cofactor redox couples, the forward electron transferreactions occur faster than the recombination reactions andtherefore, within hundreds of picoseconds, the electronreaches the primary electron acceptor, ubiquinone-10, sittingin the QA7 pocket. In the absence of exogenous electrondonors (i.e. cytochrome) the charge separated state D+QA

–

has a lifetime8 of 100 ms unless a loosely bound ubiquinone-10 molecule is present in the9 QB pocket of the enzyme whereit acts as secondary electron acceptor. The state D+QB

– ismore stable, with a lifetime of one or two seconds. In thepresence of cytochrome, the secondary quinone can allocatea second electron yielded from the absorption of a newphoton, thereby functioning as a two-electron gate [3,10].During transfer of the second electron from the primary tothe secondary quinone, protons reach the interior of theprotein [11]. Finally the quinol leaves the RC and is replacedby the oxidized quinone sitting in the membrane pool [12].

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)

Eur. J. Biochem. 270, 4595–4605 (2003) � FEBS 2003 doi:10.1046/j.1432-1033.2003.03845.x

Under saturating illumination, the photocycle time scaleis in the order ofmilliseconds. A key role in the photocycle isplayed by the exchange of the two redox forms of thequinone, between the protein interior and the bilayer. Someconsiderations regarding the exchange reaction for theoxidized quinone are made in this paper, based oninvestigations into the charge recombination reactions thattake place in purified RCs reconstituted in proteoliposomes,and in the absence of exogenous electron donors. Proteo-liposomes were selected because they can be considered agood mimicking system for the photosynthetic membrane,in which the relative amounts of enzyme and quinone canbe altered easily, in contrast to the isolated ICM, calledchromatophores, where changing quinone concentration isa laborious task [13]. Moreover, in the ICM the presence ofthe entire and active electron transport chain would requirethe use of decouplers in order to focus the RC–quinoneinteraction. A final consideration for using liposomes is thatthe solubilizing environment may play a role, particularlywhen the QB pocket is under investigation [14,15].In this work, RCs were reconstituted in phosphatidyl-

choline liposomes, which are recognized for producing thebest results in the formation of small unilamellar vesicles.The kinetics and equilibrium of the exchange between theQB pocket and the quinone pool were estimated. Thecollected data were simulated with the well-known kineticscheme of Shinkarev & Wraight [16], and the kineticconstants of the ligand uptake (kin) and release (kout)processes were inferred. The single species time evolutioninvolved in the kinetic scheme was extracted from theoutput of the numerical simulation. Recombination reac-tions were also compared to different solubilizing environ-ments such as reverse and direct micelles.

Materials and methods

Isolation of reaction centers and QB site depletion

Reaction centers were isolated from Rhodobacter sphaero-ides strain R-26.1 following the procedure illustrated byIsaacson et al. [17]. Protein purity was established using theratio of absorbance at 280 and 802 nm (A280/A802), whichwas kept below 1.3, and the ratio of absorbance at 760 and865 nm (A760/A865), whichwas equal to or lower than 1. Theaverage quinone content was 1.8 when defined by (Q/RC).Depletion of the QB site was accomplished using the

procedure of Okamura et al. [18], with the final prepara-tions exhibiting a quinone content (Q/RC) ¼ 1.05 ± 0.05as determined by the charge recombination decay. Nochanges to the photobleaching amplitude were observedupon addition of quinone.Charge recombination kinetics were recorded at 865 nm

using a kinetic spectrophotometer implemented with anHamamatsu R928 photomultiplier (Hamamatsu Photo-nics K.K., Hamamatsu City, Japan), and a Nd-Yag Laser(Quanta System, Milan, Italy) which was used for RCphotoexcitation. Data were collected onto a DigitalOscilloscope (Tektronix, Inc., TKS3052, Beaverton, OR,USA) and trace deconvolution was performed usingsoftware developed in-house. The decay traces wererecorded until complete recovery occurred followingphotobleaching. Absorbance changes were measured

taking the baseline recorded before the flash as thestarting value. Even at high quinone concentrations, thetrace deconvolution was obtained with a high correlationcoefficient (r2) using bi-exponential functions. A drift ofless than 1.5% was observed in samples illuminated by thesole measuring beam in the time range of the experiments.Each point in the data shown below is the average ofthree different liposome preparations.

Reaction center reconstitution in proteoliposomes

RC reconstitution in liposomes was accomplished followingthe procedure outlined in [19–21]. One to eight milligrams of1,2-diacyl-sn-glycero-3-phosphocholine (used at 48% pur-ity, Sigma) were dissolved in 500 lL of chloroform towhich, when needed, aliquots of a 1 mM ubiquinone-10(Sigma) solution were added. The resulting solution wascarefully dried under a stream of nitrogen in an Eppendorftube, to form an evenly distributed film of lipids. Fivehundred microlitres of a 4% (w/v) sodium cholate solution(Sigma) in phosphate buffer, pH 6.8, 100 mM KCl wereadded to the lipid film. Lipids were solubilized by 10–20repeated one-second sonications (Sonifier Mod. 250, Bran-sonUltrasonic Corporation, Danbury, CT, USA) to form ahomogenous solution. This solution was added to theQB site-depleted RC (90 lM), shaken vigorously and storedfor 15 min at 4 �C. Finally, the solution was loaded onto a15 cm Sephadex G-50 Superfine column (Pharmacia)previously equilibrated with the phosphate buffer. Theband containing RC incorporating liposomes elutes rapidly,and optical measurements were carried out. Proteolipo-somes were prepared with different quinone/RC (Q/RC)ratios while still maintaining a constant enzyme concentra-tion. The RC orientation in the liposome bilayer wasinferred from the decrease in the total amount of photo-bleaching at 865 nm before and after the addition ofreduced cytochrome c (Sigma). The two possible orienta-tions of RCs were found to be equally distributed.

Dynamic light scattering measurements. The hydro-dynamic diameter of liposomes was determined bymeans ofdynamic light scattering using a Brookhaven InstrumentsCorporation goniometer (BI-200SM) (New York, USA)equipped with a helium/neon laser source (wavelength632.8 nm). Samples were contained in cylindrical opticalcells with a diameter of 1 cm while an external thermostatmaintained the temperature at 20.0 ± 0.1 �C. All dynamiclight scattering determinations were made at a scatteringangle of 90�. Data were acquired within the 1–104 ns decaytime range that is necessary to determine the signal fromparticles.The diffusion coefficient D,30 was extracted from the

measured autocorrelation function by a cumulants method[22,23] using BI-PCSW SIMPLE CUMULANTS software (Brook-haven Instruments Corporation, New York, USA).In this method, the logarithm of the correlation function,

g(s),31 fits to a power series of the correlation time (s):

ln gðsÞf g ¼ Aþ Bs þ Cs2 þ :::

where A is a constant that depends on the instrumentsetting and

4596 F. Milano et al. (Eur. J. Biochem. 270) � FEBS 2003

B ¼ ��C ¼ DQ2

Q ¼ [4p · n · sen(Q/2)/k], with Q being the modulus ofthe scattering vector, n being the refraction index of thesolution, k being the wavelength and Q/2 being thescattering angle); and C is equal to33

1

2

Z10

ðC � �CÞ2CðCÞdC

24

35

where, C and C(C) are the decay velocity and the decayvelocity distribution, respectively). The ratio C/B2 rep-resents the size polydispersity distribution.In the hypothesis that particles behave like hard spheres

the average hydrodynamic radius (R) was calculated fromD using the Stokes–Einstein equation,

R ¼ kBT=6pgD

where g is the water viscosity, kB is the Boltzmannconstant and T is the absolute temperature.The geometry of the liposomes is in agreement with that

obtained by Palazzo et al. [24] for liposomes prepared in thesame way. Combining the parameters obtained for thepreparation of liposomes as summarized in Table 1, it ispossible to estimate a RC/liposome ratio of 500 ± 150depending on the lipid/protein ratio used to prepare theliposomes (see below). These values correspond to an RCsurface concentration ranging from 2.7 to 20.0 nmolÆm2.The lower concentration is in agreement with 3.0 nmolÆm2

calculated for chromatophores assigned a radius of 50 nm[25,26] and using the 50–60 RC/chromatophore ratio asfound by Saphon et al. [27].It is well known that the radius of liposomes is influenced

by the molar ratio of lipid/detergent in the mixed micelles36starting solution, and in our preparations this ratio wasalways below the critical value of 1.33 at which thetransition between the extended bilayer sheet and themicelle takes place. Each of the above described experimentsexhibits no significant variation in the diameter of theliposomes 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 beassumed as a reasonable37 estimate of the liposomes radius.The measurements made on both liposomes containing theRC (proteoliposomes), and pure liposomes (not containingprotein), gave substantially the same results.Reconstitution of the protein was confirmed by prepar-

ing liposomes in the presence of a fluorescent lipid(1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino]-dodecanoyl]-sn-glycero-3-phosphocholine38 purchased fromAvanti Polar Lipids Inc., Alabaster, AL, USA), andrecording the visible spectra and fluorescence of the solution

eluted from the column39 [19]. The RC elutes in a single sharpband that coincides with the lipid elution, indicating that theproteins are completely reconstituted into liposomes.40

Results and discussion

The kinetic scheme and data analysis

The reaction scheme outlined in Fig. 1 shows the kineticconstants for the final electron acceptor reactions. Thereactions take place in the neutral state (lower row), and inthe charge separated state that is generated in the RCfollowing the absorption of a photon in the absence of anexogenous electron donor (upper row). Several descriptionsof the scheme are available, the most detailed of which wasgiven by Shinkarev & Wraight [16].In the dark the RCs undergo a binding equilibrium in

which the loosely bound quinones are taken up and releasedfrom the QB site [12]. After a short light pulse, the RCsundergo a charge separation process, where an electron istransferred fromD to a primary quinone acceptor located inthe QA binding site. For proteins in which the QB pocket isempty, a charge recombination occurs with a phenomen-ological monoexponential decay constant [9] kF ¼ kAD

41which is 8 s)1 (kF is the phenomenological delay constantof the fast phase and kAD is the back electron transferconstant from the D+QA

– and D+QA–QB states). In RCs

which have the QB pocket occupied, the electron rapidlyequilibrates between the two final acceptors with an equilib-rium constant (LAB) that can be expressed as42 LAB ¼kAB/kBA (kAB being the forward electron transfer constantfromD+QA

–QB toD+QAQB– and kBA being the backward

electron transfer from D+QAQB– to D+QA

)QB). When theQB pockets are fully occupied, the charge recombinationreaction is also monoexponential, with a phenomenologicalrate constant, ks:

Table 1. 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

Fig. 1. The kinetic scheme for reaction centers in the presence of quinone

association and dissociation (quinone exchange), both in the dark and in

the charge separated state. The constants in the scheme are defined as

follows: kAD ¼ back electron transfer constant from the D+QA– and

D+QA–QB 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 con-

stant; 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 koutare assumed to be independent of the redox state of QA (see text for

discussion).

� FEBS 2003 Quinone exchange in liposomes (Eur. J. Biochem. 270) 4597

ðkAD þ kBDLABÞ1

1þ LAB

� � kAD

1

1þ LAB

� �Eqn ð1Þ

This approximation holds because the direct recombinationreaction from the D+QAQB

– state has a negligible kineticconstant (kBD < 0.1 s)1) [28,29]. In the presence of asubsaturating quinone concentration, only a fraction of theQB sites can be filled and the decay can be fitted with thesum of two exponential decays:

DAðtÞ ¼ DA0fastexpð�kFtÞ þ DA0

slow expð�kStÞ Eqn ð2Þ

where t is the time, DA(t) represents the amplitude at anyinstant t, andDAfast

0 andDAslow0 represent the amplitudes of

the fast and slow phase respectively.Proteoliposomes were prepared using QB depleted reac-

tion centers in the presence of increasing amounts ofubiquinone-10,45 the naturally occurring quinone in the QB

site. Charge recombination kinetics were recorded and timeevolution traces of absorbance changes were fitted(r2 > 0.995) using Eqn (2) where kF and kS represent thephenomenological decay constants of the fast and slowphase, respectively. In this work the kF constant is assumedequivalent to the kinetic constant kAD (8.3 s)1) of the decayfrom the QA

– containing states (Fig. 1). Indeed, uponaddition of inhibitors of QB functionality, the decay of thecharge separated state is monoexponential, with a constantslightly faster than the kF (10 s)1), indicating that thesecondary quinone is displaced from its binding site asobserved in detergent.46In contrast, kS results from more than one clear-cut

process as discussed below. As the quinone/RC ratio47increases, a rise47;48 in the slow phase amplitude and a decreasein the decay constant are observed. Figure 2 shows thedependence of the slow phase relative amplitude (titrationcurve) on the increase of Q/RC. Similarly the dependence ofthe decay constant is shown in Fig. 3.Under these conditions the binding reaction has a role in

the slow component of the charge recombination. The slow

decay constant depends both on the rate ratio between thequinone exchange and the charge recombination from thestates D+QA

– or D+QA–QB, in addition to Q/RC.

The quinone release rate kout[D+QA

)QB] can be normal-ized to the back electron transfer rate from the appropriatestate; 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) > 1 the exchange is defined as fast, whereasfor (kout/kAD) < 1 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 theinfluence played by the surroundings on kin, kout and LAB.For instance, in direct lauryl dimethyl amino N-oxide(LDAO)49 micelles a decay sum of two exponential isobserved [18] with a subsaturating quinone concentration[i.e. a fast phase with a decay constant (kF ¼ kAD) and aslow 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 thecharge recombination reaction, hence the relative amplitudeof the slow phase is proportional to the QB site occupancy.On the other hand, in direct Triton X-100 micelles a decaysum of two exponentials is observed [30] with a subsaturat-ing quinone concentration in which the kS depends on theconcentration of added quinone, ranging from 1.1 s)1 to2.7 s)1, showing a fast exchange at the QB site. Agostianoet al. [31] found that the charge separated state of RCssolubilized in phospholipid reverse micelles will decay as thesum of two exponentials. The reverse micelles are dissolvedin hexane where the unbound quinone is highly soluble. Thedecay has a kF ¼ kAD and a slow phase with a constant kSdecreasing from 3 s)1 to 1 s)1, and a relative amplitudeincreasing to 1.0 for 400 £ Q/RC £ 7000. Such behaviourwas explained in terms of fast quinone exchange.Assuming quinone molecules uniformly distributed

among vesicles of different sizes, Palazzo et al. [24] studiedthe influence of the spread of the local solute concentrationon the phenomenological kinetic constants.In the present work the Q/RC ratio ranged from 0.02 to

4, and fullQB reconstitution was obtained for values higherthan 3. The long chain exogenous quinone was confined to

Fig. 2. 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 mixedmicelles, where [RC] ¼ 8.3 lM;s, 2.1 lMRC

made up in 0.025% LDAO in 20 mM tris buffer pH 8, where the

quinone is solubilized in64 Triton X-100.

Fig. 3. Slow phase decay constant as a function of quinone/RC molar

ratio. d, liposomes; s, detergent.

4598 F. Milano et al. (Eur. J. Biochem. 270) � FEBS 2003

the liposome bilayers. Additionally, as a direct consequenceof our liposome preparation method, a solute moleculedistribution weighted by the bilayer vesicle volume wasconsidered (i.e. larger vesicles will contain larger numbers ofsolute molecules). As shown in the Appendix, under thisassumption the average local volume concentration ofquinones is the same for aggregates of all sizes and thepolydispersity can be neglected at high overall quinoneconcentration [Q]. In the investigated [Q] concentrationrange 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 withkF ¼ kAD and a slow phase kS decreasing from 1.5 s)1 to0.5 s)1. Using the asymptotic kS value in the equilibriumconstant, LAB is found to be 15.6. It should be noted thatwhen the condition (kout/kAD) > 1 occurs, the quinoneuptake and release take place during the charge recombi-nation reaction. The exchange regime and the value of someconstants for the three solubilizing environments aresummarized in Table 2.

Numerical simulations

The set of differential equations [Eqn (3)] required for thekinetic scheme shown in Fig. 1 was numerically solved by afourth order Runge–Kutta method. Using this approach avalue for the quinone uptake and release kinetic constantsand therefore the quinone binding constant (KB ¼ kin/kout)can be determined. The symbols used in Eqn. (3) are thesame as those used in [16]. Numerical simulations have beencarried out for the lipid/protein molar ratio 1000 : 1 byusing the values listed in Table 3.

dx/dt¼�ðkAD þ kinqÞxþ kouty

dy/dt¼ kinqxþ kBAz� ðkAD þ kAB þ koutÞydw/dt¼ koutuþ kADx� kinqw

dz/dt¼ kABy� ðkBA þ kBDÞzdu/dt¼kinqwþ kADyþ kBDz� koutu

dq/dt¼�kinqqðwþ xÞ þ kinðyþ uÞq

8>>>>>>>>><>>>>>>>>>:

Eqn ð3Þ

where x ¼ ½DþQ�A �=½RC�; y ¼ ½DþQ�

AQB�=½RC�; z ¼½DþQAQ�

B �=½RC�; w ¼ ½DQA�=½RC�; u ¼ ½DQAQB�=½RC�;q ¼ ½Q�free=½Q�total and q ¼½RC�=½Q�total.Immediately after the flash, at time zero, the electron is

found only in the charge separated states involving theprimary electron acceptor, i.e.D+QA andD+QA

–QB, whileQB has not yet been reached. The D+QA

–QB state rapidlydisappears, with constant kAB generating the stateD+QAQB

–, until the equilibrium is attained within fewmilliseconds. Simultaneously, the charge separated statesstart to decay and the different contributions cannot beresolved by monitoring the D+ decay. The free quinoneconcentration ([Q]free) drops from its equilibrium �dark�value and is driven to the QB site by the presence of theelectron. A typical time-evolution obtained by solvingEqn (3) is shown in Fig. 4.The quinone binding constantKB was varied in the range

1 · 105 ) 1 · 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. 5where the charged species decay is simulated for seven koutvalues at constant KB. The slow decay constant ks is weaklydependent on kout for large and small kout/kAD values,whereas the dependence increases when this ratio is closeto 1. The overall dependencies of simulated ks and DA0

slow

upon KB and kout/kAD are illustrated in Fig. 6.The kin and kout values that minimize the square-root

difference between the simulated and experimental traceswere obtained by using the �simple search method� [32] witha tolerance of 10)4 giving kin ¼ 7.2 · 107 M)1Æs)1 andkout ¼ 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 simu-

lated data for the reconstitution of QB site experiments inproteoliposomes (Fig. 7) is very satisfying.

Discussion

An important issue arising from the above experimentaland simulated data is the different behaviour of thequinone exchange when passing from direct micelles to

Table 2. Constants and exchange domains for three different solubilizing environments.

RC solubilizing environment kAD (s)1) LAB KB (M)1) kout/kAD

LDAO direct micelles 8.3 9–10a 107 <1

LDAO direct micelles [51] 8.3 20 107 <1

Triton X-100 direct micelles [30] 8.3 6–8a 5 · 107 >1

Phospholipids reverse micelles 8.3 11.5 1.2 · 104 >1

Phosphatidylcholine proteliposomesb 8.3 15 1.8 · 106 4.8

65 a Calculated from the equation (kAD/kS) ) 1 using the values kAD ¼ 8.3 s)1 and ks ¼ 0.8 s)1 [16]. b This work.

Table 3. Numerical value for the constants employed in the simulation of

D+ decay. LAB is taken from Table 2; kAB and kBD from [28] [29]; kBAis 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 Value

kAB 104 s)1

kBD 1 · 10)2 s)1

kBA 6.6 · 102 s)1

66kAD 8.3 s)1

� FEBS 2003 Quinone exchange in liposomes (Eur. J. Biochem. 270) 4599

proteoliposomes. The main difference between these twosolubilizing environments is their organization with theenzyme. RC–LDAO complexes have been characterized bysmall angle neutron scattering [33,34]. The complex isformed by a toroidally shaped group of micelles surround-ing the most hydrophobic part of the protein. In thesecomplexes the detergent around the protein is organizedwith the chain perpendicular to the protein surface and withthe terminal region sticking into the protein. This reducesthe hydrophobic portion of the detergent in which freequinone can diffuse ( 1500 A3) [33]. Crystallographic data[14] shows that the detergent itself is located in the channelinto which the quinone isoprenoid chain sits in the enzyme.This explains the slow exchange process of the quinone at itsbinding site. Conversely, the dimensions of Triton X-100micelles [35] are larger than those formed by LDAO,

thereby allowing a larger quinone pool size as well as higherligand mobility.The proteoliposomes are topologically similar to the

detergent–RC50 complexes, i.e. they are disconnected solubi-lizing environments, but they differ because proteolipo-somes can allocate a large number of proteins, in the orderof hundreds of RCs per vesicle. As a consequence, thenumber of quinones per liposome ranges from tens tohundreds, and fluctuations in the local concentration can beneglected. Liposomes can therefore be used for drawinggeneral conclusions on quinone binding at the QB site. Thelipophylic environment represented by proteoliposomes hasseveral advantages in describing the exchange of quinone inphotosynthetic membranes compared to the RC–detergentcomplexes: (a) the quinone is arranged in the bilayer in asimilar manner to chromatophores, where quinone canfreely diffuse towards and away from the enzyme, and thelarge volume of the bilayer allows the accommodation alarge number of ligands; (b) the arrangement of the lipidmolecules around the RC is not known, but it can bereliably assumed that they will not attach with their chainsinto the protein. No direct interaction with the QB site isexpected and the channel will always be accessible for thequinone exchange; (c) The absolute value of ks measured indetergent is larger than the one obtained in saturatingconditions in liposomes, indicating a relative stabilization ofQ��B . This difference in the semiquinone stability might be

associated with small detrimental changes in theQB pocket,induced by the detergent hydrophobic chains that are absentin the case of liposomes. The absence of detrimental effectsin liposomes is also confirmed by the D�þ electron nucleardouble 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 inorder to understand the same process in photosyntheticmembranes. For a bimolecular reaction of an enzymewith asmall ligand, a reasonable approximation of the frequency

Fig. 4. 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. 5. 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.

4600 F. Milano et al. (Eur. J. Biochem. 270) � FEBS 2003

of collision (fC) in the diffusion controlled regime can beobtained by simple considerations on the mobility of thetwo species [36]:

fC ¼ 4pr0ðDRC þDQÞ 10�3NA 4pr0DQ 10�3NA

Eqn ð4Þr0 is the minimum approaching distance in cm, assumedto be equal to the radius of the protein; NA is theAvogadro Number; DRC and DQ represent the diffusioncoefficients of the RC and quinone, respectively. Theapproximation in Eqn (4) is based on the large differencein the dimension of the colliding molecules. For mito-chondrial cytochrome bc1, a diffusion constant D ¼ 4.0 ·10)11 cm2Æs)1 was measured [37,38] and a similar order ofmagnitude can be expected for the RC, as both are largemembrane proteins.Several techniques have been used to measure the

ubiquinone-10 diffusion coefficient DQ. Using fluorescentquenching [39–42] the diffusion coefficient was found tospan the range 1 · 10)7 ) 5 · 10)6 cm2Æs)1. With thefluorescent recovery after photobleaching technique, a valuein the range 1 · 10)8 ) 5 · 10)8 cm2Æs)1 was obtained[37,43,44]. Electrochemical methods were also used, and avalue of (2.0 ± 0.4) · 10)8 cm2Æs)1 was obtained [45].According to Blackwell and coworkers [41,42] the DQ

obtained using the fluorescence quenching method can bedisregarded because it overestimates the actual value.Therefore, using DQ ¼ 2.0 · 10)8 cm2Æs)1 in Eqn (4), anfC value equal to 5.0 · 107 M

)1Æs)1 is obtained. It should benoted that although the collision frequency is slightlyoverestimated because Eqn (4) is valid for three-dimen-sional systems, this value remains within the accuracy ofthese considerations.Assuming the surface of the QB channel entrance to be

the area of the protein where a successful collision can takeplace [46], an estimate of the association constant can bemade. Assuming 30 A2 and 5000 A2 for the quinonemoiety, and L and M subunit surfaces in contact with thelipids respectively, a correction factor of 0.006 is obtained.As a consequence kin can be estimated to be 3 · 105 M

)1Æs)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 (seeAppendix) giving kin[L]v¢tail ¼ 3.6 · 105 M

)1Æs)1. This sug-gests that the rate limiting step for the association of the RC

Fig. 6. Three dimensional representation of (A) ks and (B) DA0slowdependence on KB and kout/kAD. For kout/kAD < 1 (slow exchange), the fraction of

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 > 1 (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.

Fig. 7. Comparison between simulated (s) and experimental values (d)

for ks (A) and DA0slow (B) as functions of Q/RC.

� FEBS 2003 Quinone exchange in liposomes (Eur. J. Biochem. 270) 4601

and quinone is the diffusion of the latter through theproteoliposomes, implying that the ligand in the bindingchannel, either taken up or released, moves at least as fast asin the bilayer; this can be expressed as (DQ)channel ‡ DQ.Assuming a random transfer for quinone, it will cover thebinding channel of length �X ( 50 A) in an average time of(1/kdiff) £ (�X 2/2DQ) 2 · 10)5 s.The average time for quinone release, obtained from the

simulations of 1/kout ¼ 25 ms, accounts for both theresidence time in the channel (1/kdiff) and for the timerequired to unbind from the pocket (1/kP):

1

kout¼ 1

kdiffþ 1

kP 1

kP¼ 25 ms Eqn ð5Þ

As a consequence, the bottleneck in the quinone releaseprocess is represented by the unbinding of the ligand fromits pocket. The value of 25 ms obtained from Eqn (5), iscomparable with that of 2 ms obtained by NMRmeasurements for systems kept in the dark in the presenceof ubiquinone-10 [47]. The results differ by one order ofmagnitude, and the discrepancy can be attributed to theassumption that the charge separated and neutral RCsexchange quinones with the same kinetics, regardless of theredox state ofQA (Fig. 1), as we assume that the presence ofthe hydrophobic tail has no influence on kP. This suggeststhat in the charge separated state the quinone release isslower than in the dark. This can be explained by invokingthe gated propeller twist imposed onQB by the presence of anegative charge on QA [48] that buries the quinone head inthe inner part of the QB pocket, thereby increasing theinteraction energy between the ligand and the binding site.In a forthcoming work the exchange kinetic dependence onthe 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 thepresence of an exogenous electron donor able to doublyreduce D+. Gerencser et al. [49] have measured the steady-state rate of cytochrome c turnover in detergent, demon-strating that at low ionic strength the reaction ofcytochrome c3+ unbinding from the RC is the rate limitingstep of the photocycle (1000 s)1 < koff < 2000 s)1). Byemploying the simulated kin ¼ 7.2 · 107 M

)1Æs)1 value, it ispossible to estimate the [Q]min at which quinone uptakeis not the rate limiting step: kin · [Q]min > 1000 s)1 �[Q]min > 14 lm, which agrees with the value of 25 lM usedin Gerencser’s work. Such [Q]min can easily be obtainedin our preparation and would give a quinone pool ofQ/RC 3, which is smaller than the average dimension ofthe quinone pool in chromatophores [50].Interestingly the structure of the QB pocket and the

quinone in the illuminated crystals [48] shows a stronginteraction between the protein residues and the quinoidmoiety of the ligand, based on the formation of hydrogenbonds. These bonds will, of course, disappear followingthe double reduction of the RC photocycle and protona-tion of the quinone; in some way driving the release of thequinol. It is quite tempting to conclude that the release ofthe quinol from the binding pocket would be faster thanthe quinone release because of the weaker interactionbetween the QB pocket and the reduced ligand. Presentlyhowever, the results only permit the setting of a lower

limit on the release rate of quinol, which will lead to aresult larger than the same rate for the oxidized form:53(kout)QH2

‡ (kout)Q. Conversely, from the hypothesis thatcytochrome turnover would be unchanged in both thevesicle and in detergent, i.e. that the unbinding of oxidizedcytochrome would remain the slowest step of the photo-cycle, the upper limit for the quinol release can be set to(kout)QH2

£ 1000 s)1.

Conclusions

By studying the charge recombination kinetics of reactioncenters incorporated into liposomes, thermodynamic andkinetic parameters have been inferred which regulate thephotosynthetic turnover of this important protein. Thesevalues, although obtained in a simpler environment, can bereasonably taken as a fair approximation to the onesactually working in the natural ICMs.The high value found for the quinone equilibrium binding

constantKB ¼ (kin/kout) ¼ 1.8 · 106 M)1, makes it possible

for the reaction centers to efficiently work with a smallquinone pool: we found that with a quinone/protein molarratio as small as three, theQB site was fully occupied. Whenthe electron reaches the QA site in a reaction center withoutthe quinone in the QB pocket, only the charge recombina-tion reaction can occur, which results in a loss of excitationenergy. However, in physiological conditions, where thequinone pool size has been estimated to be 10 or larger, thisis very unlikely to happen. It would be interesting toinvestigate similar reconstituted systems prepared withRC mutants with smaller charge recombination rate(kAD) constants which fulfil the slow exchange regime inliposomes.54

Acknowledgements

The authors are grateful to Professor E. Caponnetti and Dr Lucia

Pedone of Dipartimento di Chimica Fisica – Universita di Palermo for

performing the dynamic light scattering measurements. Thanks also to

Laszlo Nagy and Peter Maroti for helpful discussions. This work was

made possible thanks to the financial support of the Grants:

Meccanismi Molecolari della Fotosintesi (FIRB-MIUR) and Cofin –

MIUR 2002.

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Appendix

In this section thedistributionof ahighlyhydrophobic soluteamong vesicles of different sizes will be addressed, assumingvesicles to be spherical compartments with bi-layeredboundaries of negligible thickness. Moreover, the existenceof a density probability functionP(R) should also be definedas follows:P(R) dR equals the probability tofindaVRvesiclewith radius between R and R + dR. At this level ofapproximation, the overall concentration of vesicles [V] canbe calculated in terms of the lipid concentration [L] by thesurface area conservation law per unit volume:

½L�a ¼ ½V�Z

8pR2PðRÞdR ¼ ½V�8phR2i Eqn ð6Þ

a being the lipid head area. Additionally, the bilayervolume can be also estimated according to Palazzo et al.[24], as the product of the lipid number on the vesiclebilayer surface (8pR2/a) multiplied by the lipid tailvolume vtail:

mðRÞ ¼ ð8pR2=aÞ � mtail Eqn ð7Þ

The distribution of solute molecules S, among sphericalvesicles V, of different radius R, can be then described withthe following density function:

Pðn;RÞ ¼ Pðn jRÞPðRÞ Eqn ð8Þ

whereP(n,R) dR is the probability to find n solutemoleculesinside a VR vesicle (i.e. an aggregate of size between R andR + dR), and it equals the products of the probabilityP(R) dR to find a VR vesicle multiplied by the conditionalprobability P(n|R) to find n solute molecules in thisaggregate.As a consequence of Eqn (8) the average number of

solute molecules ÆNæ among vesicles of any size is obtainedby the summation over all possible solute molecule numbersand the integration over all vesicle size ranges:

hNi ¼Z X

n

nPðn jRÞPðRÞdR ¼Z

hNðRÞiPðRÞdR

Eqn ð9Þ

As shown by the previous equation, ÆNæ can also beexpressed in terms of the average numbers of solutemolecules among VR compartments: ÆN(R)æ ¼SnnP(n|R). The term ÆN(R)æ can also estimate, in termsof macroscopic concentration, the ratio between thebulk concentration of S molecules contained in VR

vesicles ([SR]), divided by the bulk concentration of theseaggregates [VR]:

hNðRÞi ¼ ½SR�½VR�

Eqn ð10Þ

[VR] is directly linked to the overall vesicle bulkconcentration [V] by the equation [VR] ¼ [V]P(R) dR,whereas different hypotheses can be found on therelationship between [SR] and [S], depending on theexperimental preparation method. Herein two mainassumptions will be considered: (a) the solute moleculedistribution is independent of the vesicle radius, (b) the

4604 F. Milano et al. (Eur. J. Biochem. 270) � FEBS 2003

solute molecule distribution is weighted on the volumeof 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) oneobtains:

hNðRÞi ¼ ½S�½V�

� �¼ ½S�

½L�

� �8phR2i

a

� �Eqn ð11Þ

therefore ÆN(R)æ is independent of the specific vesicle radiusR. It will however, depend on the second moment of theP(R) probability density function: ÆR2æ ¼ ÆRæ2 + r2. Infact, at fixed [L] if the average size of vesicles increases thentheir overall concentration [V] must decrease and ÆN(R)æmust increase. On the other hand, in the case of the bilayervolume weighted distribution, the concentration [SR] willresult:

½SR� ¼ ½S� mðRÞPðRÞdRRmðRÞPðRÞdR

� �¼ ½S� R2

hR2i

� �ðPðRÞdRÞ

and by means of Eqns (10 and 6) the average number ofsolute molecules in R sized vesicles will be:

hNðRÞi ¼ ½S�½L�

� �8pR2

a

� �Eqn ð12Þ

in this case ÆN(R)æ is proportional to the bilayer vesiclevolume.Defining the solute vesicle volume concentration as the

mole number of solute molecules in a vesicle divided by thebilayer volume: (SR)V ¼ n/(NAv(R)), we can now calculatethe average Æ(S)Væ:

hðSÞVi ¼�Z X

n

n

NAvðRÞ

�ðPðn jRÞPðRÞdRÞ

¼Z hNðRÞi

NAvðRÞ

� �ðPðRÞdRÞ

NA being the Avogadro number.In scenario (a), by using Eqns (11, 6 and 7) one obtains:

hðSÞVi ¼½S�½L�

1

v0tail

� �ðhR2iÞ

ZPðRÞdR

R2

� �

½S�½L�

1

v0tail

� �hR2ihRi2

!Eqn ð13Þ

where v¢tail ¼ vtailNA, as obtained by Palazzo et al. [24].On the other hand, in scenario (b) ÆN(R)æ can be

calculated using Eqns (12, 6 and 7):

hNðRÞi ¼ ½S�½V�

� �R2

hRi2

!¼ ½S�

½L�

� �8pR2

a

� �Eqn ð14Þ

and the average solute concentration will result:

hðSÞVi ¼ ½S�½L�

� �1

m0tail

� �Eqn ð15Þ

The previous equation clearly shows that in the case of asolute distribution weighted on the bilayer volume, theaverage vesicle volume concentration of solute Æ(S)Væ isindependent of the vesicle size, and the experimental resultsfrom different vesicle size distributions can be directlycompared.Moreover, at fixed lipid concentration [L], Æ(S)Væis proportional to the bulk solute concentration [S] and thisallows one to use this value in the kinetic equations, keepingin mind that the bimolecular kinetic constants used inEqn (8) must be corrected multiplied by [L]v¢tail to obtainthe real constants.Another important point is to estimate the standard

deviation of Æ(S)Væ and this can be done by first calculating:

hðSÞ2Vi ¼Z X

n

n

NAmðRÞ

� �2 !

� ðPðnjRÞPðRÞdRÞ ¼ ðhðSÞVi2Þ

þ ½S�½L�

� �a

8pm02tail

� � �1

R2

�� �

and then obtaining the polydispersity index:

PhSi ¼�hS2ihSi2

�� 1 ¼ ½L�

½S�

� �a8p

� � 1

R2

� �� �

½L�½S�

� �a8p

� � 1

hR2i

� �

whilst keeping in mind Eqn (15). PÆSæ shows that byincreasing the bulk solute concentration or the averageradius of liposomes, the (S) polydispersity decreases.In the studied case the RC concentration gives:

[RC]

molÆL)1Æ(RC)VæmolÆL)1

rÆ(RC)æ

molÆL)1 PÆ(RC)æ67

8.3 · 10)6 1.5 · 10)3 7.1 · 10)5 2.2 · 10)3

whereas for the quinone we obtain:

[Q]totalmolÆL)1

Æ(Q)VæmolÆL)1

rÆ(Q)æ

molÆL)1 PÆ(Q)æ

1.5 · 10)7 2.7 · 10)5 9.5 · 10)6 1.2 · 10)1

3.1 · 10)5 5.6 · 10)3 1.3 · 10)4 5.9 · 10)4

showing that only at very low concentration ofquinones, the spread of the concentration distributionbecomes not negligible.

� FEBS 2003 Quinone exchange in liposomes (Eur. J. Biochem. 270) 4605