Investigation on the detergent role in the function of secondary quinone in bacterial reaction...

Investigation on the detergent role in the function of secondary quinone inbacterial reaction centers

Angela Agostiano1, Francesco Milano1 and Massimo Trotta2

1Dipartimento di Chimica, UniversitaÁ di Bari, Italy; 2Centro Studi Chimico Fisici sull'Interazione Luce Materia, UniversitaÁ di Bari, Italy

In this paper are reported studies on the detergent role in isolated reaction centers (RC) from Rhodobacter

sphaeroides, over a large range of lauryldimethylamino-N-oxide (LDAO) concentrations, in influencing the

thermodynamics of the quinone exchange reaction as well as the protein aggregation. The occurrence of the quinone

exchange reaction between the QB-binding site (where QB is the second quinone molecule of two in the RC) and the

ubiquinone 0 dissolved in the different environments (water, LDAO micelles and detergent phase of the protein±

detergent complex) has also been analyzed. Measurements carried out in QB-depleted RC to which exogenous

quinone has been added show that the relative amplitudes of the slow and fast phase of the recombination reaction

depend on this parameter. The overall amount of the restored QB-functionality is affected by the concentration of

the LDAO in solution. Interpolation of the titration curves with a quadratic function obtained by simple

considerations allowed the binding constant of UQ0 to the QB-binding site to be calculated. From the fitting

procedure, the distribution of the quinone in the different environments present in solution was evaluated, indicating

that the exchange reaction can take place only between the QB-site and the detergent phase. The dependence of the

quinone pool size upon the volume of the phase in which the interacting quinone is solubilized is also discussed. The

increasing difficulty in saturating the QB-pocket above the LDAO critical micellar concentration is finally related to

the association of protein±detergent complexes to form large protein clusters.

Keywords: bacterial photosynthesis; reaction center; detergents; quinone, electron transfer.

The photosynthetic reaction center (RC), an integral transmem-brane pigment±protein complex, represents an extremelyefficient molecular machine able to perform charge separationacross the membrane. The charge separation is impinged byabsorption of a photon with a quantum yield close to unity. Inpurple nonsulfur bacteria, this process is achieved through aseries of electron transfer reactions, from a bacteriochlorophyllspecial pair (D) to a primary ubiquinone acceptor (QA). Thecharge separated state D+Q2

A is stabilized by replacement of theelectron on D+ by reduced cytochrome c2 followed by a forwardelectron transfer to a secondary ubiquinone molecule (QB). Thelatter quinone is loosely bound to a relatively polar proteindomain from which it can freely exchange with a membranequinone pool.

Because of its hydrophobicity, the RC is solubilized andpurified by using detergents to disrupt the membrane and keep itin solution forming a protein±detergent complex[ 1]. RCs fromRhodopseudomonas viridis and Rhodobacter sphaeroides havebeen extensively studied and the three-dimensional structure ofthe protein±detergent complex is known for both the ordered

protein portion [2±4] and for the disordered detergent part [4,6].The complex has a core formed by the protein surrounded by aring-shaped micelle that spans the RC for the length of thetransmembrane a-helices, occupying the corresponding portionof the protein naturally embedded in the membrane. The roleplayed by the detergent goes further than the solubilization ofthe protein. Comparative EPR and electron nuclear doubleresonance (ENDOR) studies [7] on the electronic structure ofthe radical cation of the primary electron donor in four differentspecies of purple bacteria clearly indicate that the surfactantenvironment of the RCs influences both the electronic structureof the cofactors and the electron transfer rate. Two distinctconformations of oxidized primary donor have been observed,depending on the detergent properties and the detergent/RC ratio[8]. Global analysis of time-resolved difference spectra [9]indicated that the rate of charge separation in membrane-boundRCs is slightly slower than that of detergent solubilized RCsfrom the same strain. The detergent interacts with the channeltrough, in which the loosely bound ubiquinone 10 (UQ10) at theQB-site is taken up or released [10] in its reduced form duringthe photocycle of the RC. In detergent-free RCs the secondaryelectron transfer from QA to QB was inhibited, and restored uponaddition of detergent. A study of the interaction betweenquinone and the detergent with the RC has recently beenpublished [11], in which the amount of quinone bound to the RChas been varied, in turn varying the surfactant concentration.The average number of quinones per RC micelles is shown todetermine the time of the slow component of D+ dark relaxation.The apparent dependence of the secondary acceptor functioningupon environmental parameters is interpreted in view of theevidence that the complete saturation of the QB binding site isdifficult to achieve in detergent-solubilized RCs for highly

Eur. J. Biochem. 262, 358±364 (1999) q FEBS 1999

Correspondence to M. Trotta, Centro Studi Chimico Fisici sull'Interazione

Luce Materia, c/o Dept of Chemistry, University of Bari, Via Orabona 4,

I-70126 Bari, Italy. Fax: +39 08 5442128, Tel: +39 08 5442028,

E-mail: [email protected]

Abbreviations: LDAO, lauryldimethylamino-N-oxide; RC, reaction center;

QB, secondary ubiquinone binding site; UQ0, ubiquinone 0; UQ10,

ubiquinone 10; cmc, critical micellar concentration; ENDOR, electron

nuclear double resonance;

Note: dedicated to Professor Americo Inglese (18 April 1946±20 November

1998)

(Received 25 January 1999, accepted 25 February 1999)

q FEBS 1999 Secondary quinone function in bacterial reaction centers (Eur. J. Biochem. 262) 359

hydrophobic quinones. In this work we addressed the roleplayed by the RC-bound detergent on the quinone exchangeequilibrium at the QB-site for a smaller water-soluble homolog,ubiquinone 0 (UQ0). The results of this work allowed us toobtain an equilibrium constant value for the exchange reactionthat takes place between the binding site and the detergentportion of the complex. Furthermore, the experiments performedin the presence of detergent micelles not bound to the proteinindicate that protein±detergent complexes tend to aggregate insmall clusters that interact with the exchangeable quinone in adifferent manner to the isolated units.

MATERIALS AND METHODS

Chemicals

Commercial 30% lauryldimethylamino-N-oxide (LDAO) solu-tion was obtained from Fluka and its exact concentrationwas assessed by densitometric measurements [12,13]. 2,3-dimethoxy-5-methyl-1,4-p-benzoquinone (UQ0) was obtainedfrom Sigma and used without any further purification. Allmeasurements were performed at pH 8.1, buffered with 10 mmTris from Sigma in presence of 1 mm of EDTA from Aldrich.

Reaction centers

RCs from the carotenoidless strain R. sphaeroides R26 weregrown photosynthetically and isolated following the methodillustrated by Feher and Okamura [14]. One-quinone RCs wereprepared by the method of Okamura [15] and the quinonecontent was optically assessed from the kinetics of donor D+

recovery after a light pulse [16]. In all preparations, the residualQB-content was lower than 10%. Reconstitution of QB with UQ0

was performed by adding solid quinone to the solution.The equilibration of the RC with the chosen concentration

of LDAO was performed using combined ion-exchange/gel-filtration chromatography [17]. The RCs were initially loadedon the column where they stopped at the ion-exchange layer(DEAE-52, Whatman). The column was then equilibrated withthe buffer T10LXE1, where X represents the percentage of LDAOfor 2±3 h using a flow of 5 mL´min21. Finally, after a pulse of300 mm NaCl the RC passed onto the gel-filtrating layer(Sephadex G-15, Pharmacia) and was then collected in a singlefraction. It should be noted that the samples were salt free andthat the eluting peak became broader as the LDAO concentrationincreased. The minimum suitable concentration of LDAO is0.0125%, below which the RCs precipitate in the column.

The optical spectra of the RCs were recorded before and aftereach titration experiment, and no major changes in the nearinfrared zone (600±1000 nm) were observed. Wavelengthsbelow 600 nm were not useful due to the strong absorbtion ofthe quinone.

Optical measurements

Absorbtion measurements were performed on a Jasco (UV-VisMod. 7800) spectrophotometer. The flash-induced photobleach-ing experiments were performed on a single beam spectro-photometer of local design. The excitation was accomplished bya Xenon flash lamp (Hamamatsu) with a pulse length of<100 ms, well below the time constant of the fastest recordedprocess (100 ms). The rapid changes in the optical absorbancewere collected on a R928 photomultiplier (Hamamatsu) whoseoutput, after filtering and amplification, was recorded on a12-bit Keytley DAS-1202 board and the acquired data stored on

a computer. The fitting on the kinetic traces was performedusing a nonlinear least square fitting algorithm [18] imple-mented on locally developed software.

RESULTS

The optical traces of the charge recombination reactionsrecorded at 860 nm are shown in Fig. 1 for QB-depleted RCsto which the exogenous water soluble UQ0 has been added. Alltraces exhibit a biexponential behavior, with rate constantsrelative to the fast and the slow phases equal to 10 and 1 s21,respectively. On increasing the quinone concentration, theoverall kinetic of D+ recovery slows down, passing from analmost pure D+Q2

A recombination (trace A, with fast phasegreater than 85%) to a substantially pure D+QAQ2

B recombina-tion (trace C).

The QB-site occupancy therefore increases with the increaseof the external quinone concentration and the relative ampli-tudes of the slow and the fast phases depend on this parameter.Figure 2A illustrates the fraction of slow phase (F) as functionof the total quinone concentration in solution; the curve shows asigmoidal shape as expected for the titration of the QB-site. Thesame kind of experiment has been performed at variousdetergent concentrations chosen in the range 0.5±10 mm,below and above the LDAO critical micellar concentration(cmc) of 1 mm [19,20]. The total amount of photooxidizeddimer (initial amplitude) and the main features of the titrationcurves are retained throughout the entire range of detergent

Fig. 1. Repopulation of the QB site with UQ0. Changes in the optical

absorbance recorded at 860 nm for a 3-mm solution of RC at in presence of

0.025% of LDAO. Trace (a), QB-depleted RC; trace (b), partially

reconstituted QB-site (UQ0 200 mm); trace (c), fully reconstituted QB-site

(UQ0 1 mm).

360 A. Agostiano et al. (Eur. J. Biochem. 262) q FEBS 1999

concentrations, although the addition of increasing amounts ofdetergent results in the stretching of the ramp of the sigmoid(Fig. 2B). The plateau of the curves is reached at a quinoneconcentration that increases with the raising of LDAOconcentration from 200 mm to approximately 1 mm. The overallamount of restored QB-functionality is affected by theconcentration of LDAO in solution (Fig. 3); below the LDAOcmc, more than 90% the QB-functionality is recovered; abovethe cmc this value is reduced to less than 70%. In contrast, anyattempt to follow the restoration of the QB-functionality byadding the native UQ10 was unsuccessful, due to the difficulty ofadministering known amounts of quinone into solution withoutusing solvents. We performed the reconstitution experiment atconstant UQ10 concentration, corresponding to its solubility insolution, and found that the fraction of slow phase wasindependent of LDAO concentration.

DISCUSSION

The data shown in Fig. 2 indicate the occurrence of a bindingreaction coupled with electron transfer at the QB-site for theexogenous quinone, as illustrated in the kinetic scheme shown inFig. 3. These titration curves have been interpolated with aquadratic function obtained by simple considerations on themass balance for the quinone distributed in the different phases:

�Q�T � �Q�H2O 1 �Q�RCm 1 �Q�B 1 �Q�Dm �1�

where [Q]T = total added quinine, [Q]H20= quinone in aqueous

phase, [Q]RCm = quinone in RC micelle, [Q]B = quinone in thebinding site and [Q]Dm = quinone in pure detergent micelles.Similarly a mass balance can be written for the RC±detergentcomplexes:

�RC�T � �DQA� 1 �Q�B 1 �RC�NA 1 �RC�O �2�

where [RC]T = total RC, [DQA] = RC QB-depleted, [RC]NA = RCnot accessible, [RC]O = RC with QB-site populated with UQ10

and concentrations are referred to water.The three independent equilibrium constant for all the phases

present in solution are the binding constant of the quinone at theQB-site and the quinone partition constants for the protein±detergent complex and for the micelles, given by the Eqns 3±5,respectively:

K 21D �

�Q�RCm�DQA�

�Q�B�3�

KRCm ��Q�RCm 1 �Q�B�

�Q�H2O_V �RC�T

�4�

KDm ��Q�Dm�

�Q�H2O~V �Dm�

�5�

where K 21D = dissociation constant of quinone at QB site,

KRCm = quinone patition constant of water : RC micelles,KDm = quinone partition constant of water : pure micelles,VÇ = partial molar volume of RC micelle, VÄ = partial molarvolume of pure detergent micelle and [Dm] = pure detergentmicelle concentration. The partition constants are referred to thevolume of the solution.

The fraction F of populated QB functionality, obtained bysolving Eqns 1±5, gives a classical quadratic Eqn 6 with K 21

D ,KRCm, [RC]NA and [RC]O adjustable parameters:

F �b 2

��b2 2 4ac

p2a

�6�

where

a � �RC�T 1 11

KRCm_V �RC�T

�1 1 KDm~V �Dm��

� ��7�

b � �Q�T 1 ��RC�T 2 �RC�NA 1 KD 1 �RC�O�

£ 1 11

KRCm_V �RC�T

�1 1 KDm~V �Dm��

� ��8�

c � �Q�T 1 2�RC�NA

�RC�T

� �1 ��RC�T 2 �RC�NA 1 KD 1 �RC�O�

£ 1 11

KRCm_V �RC�T

�1 1 KDm~V �Dm��

� ��9�

The results of the fitting procedure are summarized in Table 1.The average value of 89 for KRCm obtained as a fit parameter

agrees well with the value of 60 for the partition constantbetween pure detergent micelles and water (KDm) obtained fromdilution enthalpy measurements [21]. The smaller value of KDm

Fig. 2. QB-site reconstitution. Slow phase fraction against overall quinone

concentration for (X) 0.025% LDAO and (W) 0.2% LDAO.

Fig. 3. Recovery of the QB-functionality as function of the LDAO

concentration in solution. The arrow indicates the LDAO cmc value.


compared to KRCm is reasonable due the fact that in the latterconstant, together with the affinity of the quinone for the`organic phase' represented by the micelles, the term related tothe affinity toward the binding site has to be taken into account.The solubility of quinone in the protein±detergent complex willtherefore increase compared to solubility observed in puredetergent micelles. The logarithm of this value (1.78) is twicethat reported in literature for the octanol/water system (0.78)[22] indicating that the tendency of UQ0 to associate with LDAOmicelles cannot be predicted using octanol as the referencesolvent.

Figure 4A shows the distribution of quinone in the differentenvironments present in solution, as results from the fit. It can beclearly seen that the quinone solubilized in the micellar phase(with or without protein) represents only a very small fraction ofthe total. In Fig. 5B, the ratios of the quinone in the differentphases and the RC concentration are reported. From these data,the saturation of the QB-binding site and the pool-size dimen-sions can be related to the overall quinone concentration.

An approximate value for the binding constant of UQ0 to theQB binding site at different LDAO concentration can be inferredfrom the reciprocal of the quinone concentration at which half ofthe slow phase has been recovered. These values are in verygood agreement with those previously reported [23], and areshown in Table 2. Their similarity at different detergentconcentrations indicates that the thermodynamics of the processare not altered substantially by the detergent. The approximatevalues of the binding constant inferred from the titration curvesare not very different from those obtained by the exact solutionof the kinetic scheme for the exchangeable quinone, followingthe model by Wright and Shinkarev (K 1

bind) [24], as well as fromthose calculated by our fitting procedure (K mod

bind ), reported incolumns 2 and 3 of Table 2. This is not surprising, as our systemfails in the `slow exchange' category in which the in and outreactions of the quinone binding are both slower than the slowestcharge recombination reactions [25]. Under this assumption, an

electron exchange equilibrium constant LAB < 10 for thereaction (Scheme 1):

D�Q2A QB , D�QAQ2

B

can be obtained, very close to those given in literature [26](Fig. 6).

The three reported methods give values for the bindingconstant referred to bulk quantities. These values can be

Table 1. Parameters relative to the fitting procedure for eqn 6.

Abbreviations are explained in the text.

LDAO % [RC]/mm KRCm [RC]NA [RC]O

% of

[RC]NA [Dm]

00125 2�.47 84�.9 0�.0341 0�.001 13�.8 ±

0�.0167 2�.77 110�.3 0�.445 0�.283 16�.1 ±

0�.0208 2�.59 126�.0 0�.284 0�.068 11�.0 ±

0�.0250 2�.96 84�.3 0�.399 0�.190 13�.5 ±

0�.0502 3�.15 65�.8 0�.660 0�.240 21�.0 15�.0

0�.100 3�.02 83�.4 0�.721 0�.376 23�.9 44�.5

0�.200 2�.95 70�.8 0�.855 0�.279 29�.0 105�.0

Fig. 4. Kinetic scheme for the electron transfer and quinone exchange

reactions.

Fig. 5. Distribution of quinone in different phases. (A) Concentration of

quinone as obtained from the fit: solid line [Q]T, dotted line [Q]H2O, dot-

dashed line [Q]Dm, dashed line [Q]RCm. (B) Amount of quinone in the

micellar phase per RC: solid line [Q]RCm + [Q]B, dotted line [Q]RCm, dot-

dashed line [Q]Dm, dashed line [Q]B.

Table 2. Summary of the binding constant obtained from different

approaches. Kbind, binding constant obtained graphically; K 1bind, binding

constant obtained from the model of Wright and Shinkarev [24]; K modbind ,

binding constant obtained from our model. Pool size is that of the pool size

at maximum QB repopulation

LDAO

Kbind

(mm21)

K 1bind

(mm21)

K modbind

(mm21) Pool size

0�.0125 10�.9 13�.4 14�.1 1.10 �^ 0.19

0�.0167 16�.1 18�.7 18�.5 1.10 �^ 0.19

0�.0208 14�.7 14�.6 17 1.10 �^ 0.19

0�.0250 11�.8 16�.4 13�.2 1.10 �^ 0.19

0�.0500 8�.7 8�.3 12�.2 2.30 �^ 0.41

0�.100 10�.1 11�.3 14�.9 3.70 �^ 0.64

0�.200 7�.3 8�.1 15�.2 4.60 �^ 0.81


transformed into the quinone pool size, the number of quinonesper RC, using the local concentration of the quinone and thepartition constant previously reported.

Under the assumption that only the quinone dissolved in themicellar phase interacts with the QB-site complex, it is possibleto calculate the size of the quinone pool at which the RCpresents half of the binding sites occupied:

Pool size �KRCm�Q�H2O�RC�T _V

�RC�T�10�

where VÇ = 51.2 ^ 9 dm3´mol21. The size of the quinone poolfor UQ0 ranges between 0.3±0.4. Similarly, it is possible todefine the pool size needed for populating half of the QB-binding site with the inhibitors as well as for other quinones that,because of their higher hydrophobicity, can be considered to bedissolved in the sole micellar phase. From the data in literature[27] the value for the inhibitor pool size of terbutryne drops inthe order of 1±10 £ 1026 molecules per RC. For azido-atrazine,this value grows to 1±10 £ 1024. In the case of ubiquinoneswith different lengths of isoprenoid tail, the pool size rangesfrom 5 £ 1024 in the case of UQ1 to 5 £ 1026 when using UQ5.

A value of 120 mm21 has been reported for the bindingconstant for UQ10 in RCs dissolved in reverse micelles inhexane [28]. From such value, a pool size of <3±4000 isinferred, taking into consideration that in this case the lipo-soluble molecule interacts with the protein directly from thebulk of the solution. A comparison between the reverse micellesand the water systems, although different, can be useful to gaininformation on the quinone±RC interaction in photosyntheticmembrane. Indeed, in the case studied in the present paper(protein/detergent/UQ0 in water) the quinone interacting withthe RC is confined in a limited portion of space, similar to thenative UQ10 in the chromatophors, while in the case of theorganic solvent (protein/detergent/UQ10 in hexane) the ligandfreely moves in the entire volume of solution, differently fromphysiological conditions. The difference in the dimension of the

quinone pool needed to saturate the QB-site can be ascribed thento the difference in the probability that an interaction takes placebetween ligand and protein either in entering or leaving thebinding site.

All the binding constants of the detergent/protein/quinonesystem reported up to now are based upon the assumption of theidentity of total quinone concentration and the quinoneinteracting with the binding site. A more realistic model shouldconsider that if the exchange reaction takes place between thequinone solubilized into the detergent surrounding the proteinand the QB-site, as a result the interacting phase is restricted tothe micellar phase excluding any involvement of the quinonedissolved into water. By simple geometrical considerations, theprobability for an efficient interaction between the quinone andthe protein can be calculated in two different scenarios,considering the quinone inside the protein±detergent complexand the quinone in the bulk aqueous solution.

The probability that the quinone solubilized in the complexgets bound to the QB-binding site can be related, through aconstant k 0, to the self-diffusion coefficient for the quinoneinside the RC micelles DRCm and the ratio Veff/VT between theeffective volume where the interaction can take place (Veff) andthe total micelle volume:

pRCm � k 0�Q�RCmDRCm

Veff

VT

�11�

Veff can be pictured as the projection of the QB-site entrancecavity in the in the volume of the micelle (Fig. 7). We canassume:

Veff

VT

�AUQ0

dmicFC

VT

� aIFC �12�

where AUQ0 is the planar area of the quinone moiety of theubiquinones, dmic is the thickness of the micelle correspondingto the length of an LDAO molecule and FC a corrective factorthat accounts for the larger dimension of the cavity in respect tothe quinone molecule.

The probability that the quinone solubilized in the aqueousphase binds directly to the QB-binding site can be instead

Fig. 6. Schematic representation of the RC±detergent complex. The

protein portion is shown as a cylinder indicating the hydrophobic core (LM

subunits) bottomed with an half sphere for the globular part hydrophilic part

(H subunit). The channel trough which the quinone is exchanged with the QB

binding site has a projection on the protein surface whose dimension (Aeff)

correspond to the quinone moiety of the ubiquinones. The volume where the

direct interaction between the QB-site and the detergent phase takes place

(Veff) is formed by a cylinder with Aeff as base. Drawing is not to scale. See

text for further details.

Fig. 7. Recombination reaction constant for D+QAQB state as function

of the overall quinone concentration.


expressed as:

pH2O � k 0 0�Q�H2ODH2O

Aeff

AT

�RC�T �13�

where the total RC concentration [RC]T has to be nowconsidered because the capture of quinone takes place onlyfollowing a bimolecular reaction between the complex and thequinone itself. DH2O

is now the self-diffusion coefficient for thequinone in the water and Aeff/AT is the ratio between the externalarea of the micelle where the interaction can take place and thetotal external micelle area. Aeff can be similarly shown as theprojection of the QB-site entrance cavity on the external surfaceof the micelle:

Aeff

AT

� a IIFC �14�

From the previously made considerations on the partition of thequinone between the aqueous and the detergent phase, the ratiobetween the probabilities is given by:

pRCm

pH2O

�k 0

k 0 0

�Q�RCm

�Q�H2O

Dmic

DH2O

a I

a II� kKRCm

_VDmic

DH2O

a I

a II�15�

The term Dmic/DH2O can be assumed unitary since the valueof self-diffusion coefficient measured in phospholipid vesicles[29,30] and in solution are very close (DH2O

= 7 £ 10210

m2´s21. G. Colafemmina, personal communication). The terma I/a II is considered equal to 0.5 based on geometricalconsiderations and the term KRCmVÇ can be calculated equal to103. The value of k can be related to the difference in the amountof energy spent by the quinone reaching the QB-site from thetwo different environments. Clearly, this amount of energy islarger for a quinone that has to cross the palisade formed by thepolar heads of the detergent compared to that of a quinone foundin the detergent, directly in contact with the protein. Noassumption on the absolute value of k is made, although it can beassumed to be .1. In conclusion, the ratio PRCm/PH2O

can beconfidently considered ..1, allowing for the exclusion of anyinvolvement of the quinone dissolved in water in the bindingreaction. This model also remains valid above the cmc wherepure detergent micelles are also present. In spite of the fact thatthe binding constant for QB is unaltered by the amount of LDAOused for solubilizing the RC, the titration curves and the fittingparameters show that the detergent plays a role in both, varyingthe amount of restored QB-functionality (Fig. 3) and the quinonepool size necessary to saturate all the available QB-sites(Table 2). The increase of the pool size with LDAO concentra-tion, up to a value of <4, fits reasonably well with thedimension of 5±40 measured in photosynthetic membranes [31].Above the cmc, the increase in the pool size indicates that thevolume of the detergent phase in the complex is increasedbecause more LDAO has aggregated to the protein. The datareported in Fig. 3 do not show the previously reported reductionof the QB-functionality below the cmc [32]. The differentprocedure used for sample preparation could account for thisdiscrepancy. In our measurements, equilibration of the RC withthe chosen concentration of LDAO was always achieved bychromatographic conditioning (see Materials and methods),whereas a suspension of RC molecules, below the cmc, was usedto collect the photoactivity data reported by Gast et al. [32].

In our system the increasing difficulties in saturating the QB

pocket even at high UQ0 concentrations could indicate that,above the cmc, on increasing the LDAO concentration anincreasing amount of the QB-binding site remains inaccessible tothe micelle-solubilized quinone. We can therefore assume thatpure LDAO micelles and protein±detergent complexes interact,forming protein clusters in which more than one protein is

embedded in a larger detergent phase and the number ofclustered proteins increases in line with a rise in availableLDAO. The existence of RC clusters has been demonstrated[33,34] via SANS spectroscopy. At low ionic strength and abovethe LDAO cmc, particles of protein±detergent complexes with alinear dimension larger than 500 AÊ (10 times larger than thelinear dimension of a monomeric protein/detergent unit) havebeen identified. The aggregation of RC at high LDAOconcentrations is also responsible for the formation of theso-called floating pellet formed during the protein isolationprocedure [13]. In these clusters, the presence of nonactiveQB-sites can be ascribed to RCs in which the QB-site entrance isexposed to a second protein instead of being exposed to thedetergent phase. The amount of these QB-inactive RCs rises withthe increase of the cluster dimensions. As further qualitativeconfirmation of the aggregation in presence of excess detergent,the effect on QB-site reconstitution of high ionic strengthsolution has been investigated by adding NaCl (Table 3). Thepresence of high salt concentration resulted in an increasedrepopulation of the QB-site, shown by the larger amount of slowphase. This indicates that the electrostatic effect disrupts theclusters, separating them into smaller units or even into singleprotein±detergent complexes [35]. This could also explain theobservation made in our photochemical experiments of anincreased Signal-to-Noise ratio when measurements are performedin presence of high salt concentration.

Conclusions

The data discussed in this paper indicate the detergent inisolated RCs plays a role both in the accessibility of the QB

binding sites to the UQ0, as well as in the protein aggregation.Evidence that the exchange reaction takes place only betweenthe QB-site and the detergent phase, excluding any role for thequinone solubilized in the bulk of the water phase, have alsobeen reported, suggesting a certain analogy between the inter-action of the QB-site with the detergent and with the phos-pholipids in the photosynthetic membrane. Instead, the volumeof the phase in which the interacting quinone is solubilizedappears to play a role, as the size of the quinone pool depends onit. The large variability in the pool size reported for thephotosynthetic bacteria may result from the fact that thesevalues should be normalized to the concentration of RC in themembrane, i.e. to the volume of membrane available per protein.

Finally, when evaluating experiments devoted to the evalu-ation of the kinetic constant KQB for the quinone association step[36] it should be considered that, whenever using a water-soluble quinone, its partition effect may introduce a furtherexchange equilibrium between the aqueous and detergent phases.

ACKNOWLEDGEMENTS

The authors wish to thank Dr Fabio Mavelli for useful discussion. This work

has been supported by MURST (Cofin 97: Struttura, proprietaÁ e dinamica di

sistemi di interesse biologico).

Table 3. Relative amounts of slow and fast phase for the recombination

reaction at different ionic strength. [RC] = 1 mm; LDAO = 0.1%.

% of

slow phase

% of

fast phase

QB-depleted RC 12 88

+ UQ0 (1.2 mm) 74 26

+ 0�.42 m NaCl 86 14

+ 0�.86 m NaCl 88 12


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Investigation on the detergent role in the function of secondary quinone in bacterial reaction...

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