Eur J Biochem 269, 1096–1108 (2002) Ó FEBS 2002 Exploring the primary electron acceptor (QA)-site of the bacterial reaction center from Rhodobacter sphaeroides Binding mode of vitamin K derivatives Oliver Hucke, Ralf Schmid and Andreas Labahn Institut fur Physikalische Chemie, Albert-Ludwigs-Universitaăt Freiburg, Germany ă The functional replacement of the primary ubiquinone (QA) in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides with synthetic vitamin K derivatives has provided a powerful tool to investigate the electron transfer mechanism To investigate the binding mode of these quinones to the QA binding site we have determined the binding – free energy and charge recombination rate from QA to D+ (kAD) of 29 different 1,4-naphthoquinone derivatives with systematically altered structures The most striking result was that none of the eight tested compounds carrying methyl groups in both positions and of the aromatic ring exhibited functional binding To understand the binding properties of these quinones on a molecular level, the structures of the reaction center-naphthoquinone complexes were predicted with ligand docking calculations All protein– ligand structures show hydrogen bonds between the carbonyl oxygens of the quinone and AlaM260 and HisM219 as found for the native ubiquinone-10 in the X-ray structure The center-to-center distance between the naphthoquinones at QA and the native ubiquinone-10 at QB (the secondary electron acceptor) is essentially the same, compared to the native structure A detailed analysis of the docking calculations reveals that 5,8-disubstitution prohibits binding due to steric clashes of the 5-methyl group with the backbone atoms of AlaM260 and AlaM249 The experimentally determined binding free energies were reproduced with an rmsd of % kJỈmol)1 in most cases providing a valuable tool for the design of new artificial electron acceptors and inhibitors The photosynthetic reaction center (RC) of the purple bacterium Rhodobacter sphaeroides (R sphaeroides) is an intrinsic membrane protein complex that performs the conversion of light energy into chemical energy A complex framework of redox cofactors is buried in the protein matrix The cofactors are arranged in two branches, the active A-branch and the inactive B-branch, showing nearly twofold symmetry (reviewed in [1,2]) Following the absorption of a photon, an electron is transferred within 200 ps from the bacteriochlorophyll dimer, the primary donor D, via a bacteriochlorophyll monomer (BA) and a bacteriopheophytin (FA) to a tightly bound ubiquinone molecule (QA), forming the first stable – charge separated state D+QA The subsequent electron – transfer step from QA to QB proceeds on a slower time scale (% 200 ls) After rereduction of the photooxidized primary donor by a soluble cytochrome c2 the lightinduced electron transfer leads to the formation of the doubly reduced QB and concomitant binding of two protons from the cytoplasmic side of the membrane The ubiquinol dissociates from the RC and is reoxidized by the cytochrome bc1 complex releasing two protons to the periplasmic side of the membrane The net result of these reactions is a transmembrane pH difference that drives ATP synthesis In vitro, in the absence of both, an external reductant for the primary donor and the secondary quinone, the charges – on QA and D+ recombine with the rate kAD This reaction was subject to numerous spectroscopic studies One important approach to investigate this electron transfer mechanism in wild-type reaction centers was pioneered by Okamura et al [3,4] They developed a method of ubiquinone removal and readdition of ubiquinone or any other synthetic quinone Gunner et al [5] measured the temperature dependence of kAD in RCs with different anthra-, benzo- and naphthoquinone derivatives at QA Most of these compounds display a low midpoint redox potential in situ compared to the native UQ-10, leading to – an increase of the free energy difference between the D+QA and the ground state DQA Evaluating the free energy dependence of kAD Gunner et al [5] deduced that the – charge recombination from QA in native RCs is an activationless process More recently, the quinone replacement method was used to derive thermodynamic parameters for that reaction [6] and to study the forward electron Correspondence to A Labahn, Institut fur Physikalische Chemie, ă Albert-Ludwigs-Universitat Freiburg, Albertstr 23a, D-79104 ¨ Freiburg, Germany Fax: + 49 761 203 6189, Tel.: + 49 761 203 6188, E-mail: labahn@uni-freiburg.de Abbreviations: UQ-10, ubiquinone-10; NQ, 1,4-naphthoquinone; RC, reaction center; B, Blastochloris; R, Rhodobacter; QA, primary electron acceptor; QB, secondary electron acceptor; LDAO, lauryldimethylamine-N-oxide; DAD, diaminodurene Note: web page available at http://pc2-em6.physchem.uni-freiburg.de/ Andreas/homepage (Received 13 August 2001, revised 14 November 2001, accepted 23 November 2001) Keywords: ligand docking; structure activity relationship; bacterial reaction centers; Rhodobacter sphaeroides; infrared spectroscopy Ó FEBS 2002 Vitamin K derivatives at the QA-site (Eur J Biochem 269) 1097 Table Overview of the 1,4-naphthoquinone compounds used in this work The structure of the naphthoquinones is shown in Scheme Compound Quinones without undecyl tail 1,4-Naphthoquinone 2-Methyl-NQ 5-Methyl-NQ 6-Methyl-NQ 2,3-Dimethyl-NQ 2,5-Dimethyl-NQ 2,6-Dimethyl-NQ 2,7-Dimethyl-NQ 2,8-Dimethyl-NQ 5,8-Dimethyl-NQ 6,7-Dimethyl-NQ 2,3,5-Trimethyl-NQ 2,3,6-Trimethyl-NQ 2,5,8-Trimethyl-NQ 2,6,7-Trimethyl-NQ 2,3,5,8-Tetramethyl-NQ 2,3,6,7-Tetramethyl-NQ 5,6,7,8-Tetramethyl-NQ 2,5,6,7,8-Pentamethyl-NQ 2,3,5,6,7,8-Hexamethyl-NQ Quinones with undecyl tail 2-Undecyl-NQ 2-Methyl-3-undecyl-NQ 2,5-Dimethyl-3-undecyl-NQ 2,6-Dimethyl-3-undecyl-NQ 2,7-Dimethyl-3-undecyl-NQ 2,8-Dimethyl-3-undecyl-NQ 2,5,8-Trimethyl-3-undecyl-NQ 2,6,7-Trimethyl-3-undecyl-NQ 2,5,6,7,8-Pentamethyl-3-undecyl-NQ Abbreviation NQ 2MNQ 5MNQ 6MNQ 23DMNQ 25DMNQ 26DMNQ 27DMNQ 28DMNQ 58DMNQ 67DMNQ 235TMNQ 236TMNQ 258TMNQ 267TMNQ 2358TeMNQ 2367TeMNQ 5678TeMNQ 25678PMNQ HMNQ 2UNQ 2M3UNQ 25DM3UNQ 26DM3UNQ 27DM3UNQ 28DM3UNQ 258TM3UNQ 267TM3UNQ PM3UNQ ´ ´ transfer to the primary ubiquinone [7–9] Kalman & ´ Maroti used the quinone reconstitution method to study the proton binding kinetics and stoichiometry associated with the reduction of QA [10,11] It was shown that both processes are controlled by protonatable residues in the interior of the protein Measuring the delayed fluorescence of the excited dimer, D*, in RCs with different quinones as ´ QA Turzo et al [12] derived an empirical relation between the in situ free energy of the primary quinone and the charge recombination rate, providing the free energy levels of the corresponding charge separated states Similar quinone reconstitution experiments have been performed for the QB site [13–15] Palazzo et al [14,15] incorporated the reaction centers into lipid vesicles and measured the temperature dependence of the charge recombination rate – from QB to D+ Based on a detailed analysis they determined the binding free energy, enthalpy and entropy of the ubiquinone to the QB-site Methyl substituted 1,4-naphthoquinones have gained substantial interest as they bind tightly to the QA site, enabling its functional reconstitution while retaining the native ubiquinone at QB [16,17] The difference in the semiquinone anion spectra allows the direct monitoring of the electron transfer from QA to QB in the VIS region with transient absorption spectroscopy In addition, these sub- stitutions change the driving force of the electron transfer – reaction from QA to QB (and thus the electron transfer rate and equilibrium) but they affect neither conformational changes nor the protonation rates or protonation equilibria near QB Graige et al [18] and Li et al [19,20] applied this method to study the effect of the driving force on the first electron transfer to QB Similarly, the mechanism of the – – proton-coupled electron transfer reaction [QAQB+ H+ fi – QA(QBH) ] was elucidated by Okamura and coworkers They showed that this reaction is a two-step process in which fast protonation precedes rate-limiting electron transfer [17,21,22] Moreover, methyl substituted naphthoquinones play an important role in reaction centers of other photosynthetic organisms For instance, 2-methyl3-(isoprenyl)(7)9)1,4-naphthoquinone (menaquinone) was identified as the primary electron acceptor in the reaction centers from Blastochloris viridis [23], Chloroflexus aurantiacus [24] and in the photosystem I of green plants [25–27] Hence, a systematic variation of the redox potential of the naphthoquinone compounds and a detailed knowledge of their binding properties are of critical importance for the investigation of electron transfer reactions in photosynthetic reaction centers In this work, we investigated the binding properties of 29 vitamin K derivatives with respect to the QA site of the reaction center from R sphaeroides (see Table 1, Scheme 1) Their midpoint redox potentials were altered by varying both, the number and the position of methyl groups at the ring system In some cases, additionally a hydrocarbon tail was introduced in position to improve the binding affinity to QA in analogy to ubiquinone [28] Light-induced FTIR difference spectroscopy was used to detect structural changes of the binding pocket upon binding of the different quinones We measured the dissociation constants Kd of these compounds and compared our results with those from ligand docking calculations The calculated structures of the quinone-protein complexes provide insights into the aspects that govern the binding of quinones to the QA site, allowing to test whether their positions at QA are identical with that of ubiquinone-10 A preliminary account of this work has been presented elsewhere [29] MATERIALS AND METHODS Quinone-depleted reaction centers Reaction centers from the strain R sphaeroides R-26 were isolated and purified in lauryldimethylamine-N-oxide (LDAO) from photosynthetically grown cells following the procedure of Feher & Okamura [30] The ratio of absorbance (A280/A802) of the purified RCs was < 1.25 QA and QB were removed from RCs according to the method of Okamura et al [3] The residual quinone content was 0.05–0.1 mol Q per mol RC Quinones 1,4-Naphthoquinone, 2-methyl-1,4-naphthoquinone and 2-methyl-9,10-anthraquinone were purchased from Aldrich 5,6,7,8-Tetramethyl-1,4-naphthoquinone was newly synthesized from the Diels–Alder adduct of 2,3,4,5-tetramethylthiophene-1,1-dioxide and 1,4-benzoquinone according to a Ó FEBS 2002 1098 O Hucke et al (Eur J Biochem 269) binding to a population of single, noninteracting QA-sites As naphthoquinone compounds bind functionally only to the QA site [5,16] the apparent dissociation constant Kd can be obtained from Eqn (1) in case of [Q]0 ) [QRC]: DA865 ¼ Scheme The structure of the naphthoquinone compounds The rotational axis used for the description of the predicted RC-naphthoquinone complexes are indicated literature procedure [31] 5,6,7,8-Tetramethyl-1,4-naphthoquinone formed yellow needles, 1H-NMR (250 MHz, CDCl3, dH): 2.28, s, 6H; 2.53, s, 6H; 6.71, s, 2H; mp: 184– 185 °C; elemental analysis for C14H14O2 requires: C, 78.48% H, 6.57%; found: C, 78.43% H, 6.74% The synthesis of all other quinones was as described previously [31] Stock solutions of the quinones (1 lM)20 mM) were prepared in dioxan and stored at °C Determination of dissociation constants, Kd, and charge recombination rates, kAD Reconstitution of the quinones into the QA-site was accomplished by adding small amounts of the stock solution (1 lM)20 mM) to quinone-depleted RCs (20–300 nM) suspended in 10 mM Mops [3-(M-morpholino)propanesulfonic acid], 50 mM KCl, 0.04% dodecyl-b-D-maltoside at pH ¼ 7.2 The system was equilibrated for at least 25 [T ¼ (295 ± 2) (K)] Transient absorbance changes were recorded on a spectrometer of local design [32] Charge recombination kinetics were measured by monitoring the change of the donor absorbance at 865 nm following a single laser flash The rate constant kAD was obtained from a single exponential fit to the data using the software package PEAKFIT (version 4.0, SPSS Inc.) on an IBMcompatible PC The occupancy of the QA-site corresponds – to the amount of RCs in the D+QA state Its value was determined from the amplitude of the charge recombination À1 kinetics at t ¼ measured on the time scale of k AD relative to the amount of bleaching of RCs with a fully occupied QA site (see [4] for details) To account for the 5–10% RCs where ubiquinone remained at QA after quinone removal the amplitude of the signal was corrected accordingly The binding affinity of quinones to the QA-site of the reaction center can be described with the model of ligand DAmax ẵQ0 865 ẵQ0 ỵ Kd 1ị where DA865 corresponds to the concentration of bound Q at the RC, [QRC] Its value was determined from the absorbance changes of the donor recovery due to the – formation of D+QA The dissociation constant, Kd, was determined by fitting the absorbance change at 865 nm as a function of the initial quinone concentration, [Q]0 with DAmax and Kd as adjustable parameters 865 The condition of [Q]0 ) [QRC] essentially limits the applicability of the assay to Kd > 100 nM For smaller values the amplitude of the charge recombination kinetics can not be determined accurately with the experimental set up described above In this case 2-methyl-9,10-anthraquinone (0.01 mM) with the inhibition constant Ki ¼ 20 nM was used as a competitive inhibitor similar as described previously [28] This is suitable because charge recombination for this anthraquinone occurs in the microsecond range and, – hence, does not interfere with the observation of the D+QA formation of the naphthoquinones In this case the dissociation constant was determined from a two parameter least squares fit of the absorbance change vs [Q]0 according to Eqn (2): DA865 ẳ DAmax ẵQ0 865 ẵQ0 ỵ ½IŠ0 Kd =Ki ð2Þ where [I]0 is the initial concentration of the inhibitor The binding free energies Based on the work by Warncke & Dutton [33], we applied a correction method to determine the true binding free energy, DG0 , as a measure for the direct interactions between the bind quinones and the protein at the QA site The dissociation constant, Kd, is correlated to the apparent binding free energy: DG0 ẳ RT ln K1 app d 3ị where T is the temperature and R the gas constant The apparent binding free energy, DG0 , contains contribuapp tions from specific interactions between the quinone and the quinone binding site as well as unspecific hydrophobic interactions between the quinone and the nonpolar protein detergent micelles Hence, this energy can be represented as: DG0 ẳ DG0 ỵ DG0 app bind trans 4ị The transfer free energy, DG0 , describes the free energy trans change of the quinone transfer from the aqueous bulk phase to the nonpolar protein/detergent micelles DG0 can be trans approximated by the distribution of the quinone between water and an apolar solvent, e.g cyclohexane, which is given by the partition coefficient Pcw DG0 % ÀRT lnPcw trans ð5Þ Ĩ FEBS 2002 Vitamin K derivatives at the QA-site (Eur J Biochem 269) 1099 Hence, the true binding free energy, DG0 , is given by bind Eqn (6): DG0 % DG0 þ RT lnPcw bind app ð6Þ Pcw values of quinones for the system cyclohexane/water were estimated using the software package MOLECULAR MODELING PRO (Chem SW, Fairfield, CA, USA) based on the method of Ghose & Crippen [34,35] Ligand docking calculations The docking calculations were performed with FLEXX, a program designed for the docking of small to medium sized organic molecules into protein binding sites [36] During the docking procedure, the protein is considered as rigid, whereas the ligand conformation is flexible This is realized through allowing rotations around acyclic single bonds of the ligand structure Bond lengths and angles are kept constant as given in the input structure A relatively soft atom model is used by FLEXX to compensate for the rigidity of the binding site, i.e small overlap of the ligand with the receptor is tolerated by the program The docking algorithm incorporated in FLEXX is based on the chemical interactions of ligand and receptor: For the computation of ligand placements geometrically restrictive interactions are used (mainly hydrogen bonds and salt bridges) Interaction geometries were deduced from the analysis of crystallographic data The computed placements were optimized with respect to the empirical scoring function of FLEXX, which estimates the binding free energy, DG0 , of the ligand receptor complex: bind DG0 ẳ DG0 bind translat ỵ DGrot Nrot X ỵ DG0 hb fDR; Daị energy due to xation of rotation around one rotatable bond; Nrot, number of rotatable bonds) Depending on the number of possible ligand conformations, the docking calculations resulted in a set of up to % 200 possible protein ligand complexes per ligand These solutions were ranked according to the calculated binding free energy Unless stated otherwise, only the best placement (Ôplacement 1Õ), displaying the smallest value of the binding free energy, was considered for further analysis The binding site of the primary quinone in the reaction center protein from R sphaeroides was determined with the molecular modeling package WHATIF [37] based on the X-ray structure from Stowell et al [38] (RCSB PDB code 1AIJ) It included all amino acids with at least one atom ˚ lying within a distance of 6.5 A from the ubiquinone-10 molecule in the X-ray structure (a larger binding site had no effect on the results of the docking calculations) In addition, four water molecules (numbers 64, 71, 409, 410 in the PDB file), the nonheme iron atom, parts of the bacteriochlorophylls and the bacteriopheophytins located in the active branch of the reaction center are found within this cutoff distance and were therefore considered in the calculations FLEXX uses an united atom model for all nonpolar hydrogen atoms, whereas polar protons are explicitly taken into consideration Where unambiguously clear, the positions of the protons in the protein binding site were automatically assigned by FLEXX In cases of ambiguities (hydrogens of the hydroxyl groups, the N-bound proton of the histidine side chain) they were determined with WhatIf The geometries of the ligand structures were optimized with the MM+ force field of the HYPERCHEM (Hypercube Inc.) software prior to use as input files for the docking calculations neutral Hbonds X ỵ DG0 io Sample preparation for FTIR difference spectroscopy fðDR; DaÞ ionic interactions þ DG0 aro X fðDR; DaÞ arom interactions þ DG0 lipo X f à ðDRÞ ð7Þ lipoph: interactions DG0 , hb DG0 , io DG0 and DG0 are the Here, the terms aro lipo interaction energies for neutral hydrogen bonds, ionic interactions, aromatic interactions (aromatic interactions as considered by FLEXX are interactions of the electrostatic quadrupole of aromatic rings with permanent dipoles (for example of amide bonds), the quadrupoles of other aromatic rings and the induced dipoles of methyl groups) and lipophilic contacts between ligand and receptor, respectively, if ideal interaction geometries are assumed The functions f (DR,Da) and f *(DR) penalize deviations from these geometries (DR, deviation from ideal distance; Da, deviation from ideal angular geometry) The values of DG0 translat and DGrot Nrot consider the loss of translational and rotational freedom of the entire ligand molecule and the freezing of rotational degrees of freedom of the ligand structure upon binding, respectively (DG0 , loss of binding rot To reconstitute the reaction center with 1,4-naphthoquinone derivatives as primary electron acceptor, 3.5 nmol of quinone-depleted RCs were dissolved in mL buffer containing 10 mM Mops, 50 mM KCl and 0.04% dodecyl-b-D-maltoside, pH ¼ 7.0 followed by the addition of 20 lL of a 10-mM stock solution of the corresponding quinone in ethanol The samples were incubated at room temperature for a minimum period of h and then concentrated with Microcon YM-100 centrifugal filter devices (Millipore Corp., cut-off molecular mass 100 kDa) at 3000 g and °C to two aliquots of % 50 lL volume each To avoid partial denaturation of the RCs due to high ionic strength and detergent concentration upon the final concentration step, the samples were diluted by a factor of 10 with a buffer without detergent containing mM Mops and mM KCl at pH ¼ 7.0 The second concentration step yielded two highly concentrated samples of reconstituted RCs with a final volume of about 10 lL each One of them was used for FTIR difference spectroscopy (see next paragraph) whereas the remaining sample was taken to determine the occupancy of the QA-site and the charge recombination kinetics with transient absorption spectroscopy under the same conditions except that the redox mediator diaminodurene (DAD) and sodium ascorbate were omitted Ó FEBS 2002 1100 O Hucke et al (Eur J Biochem 269) FTIR difference spectroscopy FTIR difference spectroscopy was performed as described by Breton et al [39] with minor modifications The sample (% 10 lL) containing about 1.5 nmol reconstituted RC was placed onto the depression of a CaF2 window After addition of lL of the aqueous solution of the redox mediator DAD (2.5 mM) and sodium ascorbate (1.25 mM) as reductant for the primary donor, the droplet was dried under a smooth stream of nitrogen Before complete dryness, the RC film was sealed with a second CaF2 window, yielding a sample with a thickness of a few microns, minimizing the water absorption The two windows were fixed by a metal mounting and placed in the sample chamber (T ¼ ± 0.2 °C) FTIR spectroscopy was performed with a Bruker IFS 66 V/S FTIR spectrometer Light-minus-dark FTIR difference spectra were obtained by recording the spectrum of the sample under continuous illumination at a wavelength of 590 nm and subtracting the spectrum measured in the dark For each difference spectrum 1920 interferograms were accumulated To improve the signal to noise ratio, % 20 illumination cycles of the sample were averaged The FTIR difference spectra were normalized with the vector normalization method based on the spectral regions between 1500 and 1560 cm)1 and between 1670 and 1750 cm)1 The difference spectra obtained by this method (i.e in the presence of DAD/sodium ascorbate, leading to a fast reduction of the primary donor after electron transfer to QA) show exclusively the absorption changes upon the – reduction of the primary quinone, designated ÔQA/QA difference spectraÕ RESULTS Experimentally determined binding free energies The methyl substituted naphthoquinones were characterized in terms of the midpoint redox potential E1/2, charge recombination rate kAD and dissociation constant Kd (see Table 2) The in vitro midpoint redox potentials decrease with increasing number of alkyl substituents due to their inductive effect Similarly, the RCs with naphthoquinones at QA displayed low in situ redox potentials leading to an increase of the free energy difference between the states – D+QA and DQA compared to RCs with native UQ-10 as QA Therefore, the charge recombination rates kAD were significantly faster for all RCs with at least two methyl groups in the aryl ring due to the charge recombination by a thermally activated route via the – D+/A state [5,40] As previous studies revealed that even nonquinonic compounds bind to the QA site [33] indicating less specific interactions between ligand and protein, the dissociation constants of methyl substituted quinones are expected to be dominated by the quinone polarity which determines the transfer free energy However, the binding affinity is strongly affected by the substitution pattern of the aryl ring (Table 2) The most striking result of our study was obtained for all naphthoquinones with methyl groups in position and These substituents drastically weaken the association by at least a factor of 400 which represents the current limit for Kd determination To determine the actual interaction energy between quinone and RC, the apparent binding free energies were corrected for the transfer free energy (Eqn 6) by using calculated values for the corresponding partition coefficients between cyclohexane and water (Table 2) In case of the naphthoquinone compounds without an undecyl chain in position the results agree fairly well with the experimental values However, the Pcw values of the long-tail-derivatives are most likely overestimated by the Ghose–Crippen method as for each CH2 group of the aliphatic chain the same increment was added to log P The corresponding binding free energies ranged from )13.1 to +6.2 kJỈmol)1 for the compounds with an undecyl chain whereas the other quinones exhibited values from )32.0 to )21.0 kJỈmol)1 Rigid quinone binding site A main assumption in the ligand-docking calculations was that the structure of the binding pocket does not change upon binding of the different vitamin K derivatives compared to the X-ray structure determined with UQ-10 – as QA The FTIR QA/QA difference spectra show signals of both, the quinone and the adjacent region of the RC With isotope labeled ubiquinone and vitamin K1 as QA Breton et al [41] showed that the spectra in the range of 1750– 1670 and 1560–1500 cm)1 are dominated by nonquinonic contributions resulting from the response of the protein to the QA-reduction Hence, these absorption bands are indicative for the interaction between the quinone and the protein binding pocket Therefore, structural changes upon ligand binding are expected to alter specifically the vibrational frequencies and/or intensities, making FTIR difference spectroscopy an attractive method for probing the effect of the different quinones on the structure of the binding site Figure shows the QÀ =QA difference spectra obtained A for three of the naphthoquinones which are objects of this work compared to those of UQ-10 and vitamin K1 In the nonquinonic regions, the band shapes and vibrational frequencies exhibit a high similarity for all quinones No evidence for a change in the response of the protein due to – QA formation was found in these spectra supporting the assumption of a rigid binding site Docked structure with UQ-10 To test the docking algorithm of FLEXX with our system, we calculated the UQ-10/RC complex, as shown in Fig With respect to the quinone head group the calculated structure agrees very well with the X-ray structure [38] (rmsd ˚ of the head groups, 0.29 A) In contrast, the positions of the isoprenoid chains differ significantly beyond the first two isoprene units As both, the binding affinity and the midpoint redox potential of the quinone are mainly determined by the head group [28,33], these results encouraged us to extend the calculations to the naphthoquinone compounds Docked structures with naphthoquinone compounds Using our set of 29 naphthoquinone compounds (Table 1, Scheme 1) we computed the quinone-reaction center complexes For most naphthoquinones the ligand was Ó FEBS 2002 Vitamin K derivatives at the QA-site (Eur J Biochem 269) 1101 Table Comparison of the physicochemical properties of naphthoquinone compounds with different number and position of alkyl substituents in the – ring system Charge recombination rates from D+QA to DQA, kAD, were determined in one-quinone RCs monitored via the absorbance change of the rereduction of D+ following a single laser flash The dissociation constants Kd were derived from a plot of the amount of bleaching vs the initial quinone concentration according to Eqns (1,2) From an error analysis the accuracy of –log Kd was estimated to ± 0,2 Using the experimental values for Kd the apparent binding free energies, DG0 , were calculated following Eqn To account for the distribution of the quinone between app the water and the apolar protein detergent micelles the cyclohexane water partition coefficients, Pcw, were estimated with the Ghose–Crippen method [34,35] and used for correcting the apparent binding free energies (Eqn 6) The corresponding binding free energies DG0 (Exp.) were bind compared to the data from ligand docking calculations [designated DG0 (FLEXX)] The predicted complexes were analyzed in terms of the distance bind differences along the axis from the primary donor D to the primary electron acceptor QA with respect to that of 1,4-naphthoquinone [Dr(NQ)] Experimental conditions: 20–300 nM quinone-depleted RCs, 30 nM–100 lM naphthoquinone, 10 mM Mops, 50 mM KCl, 0.04% dodecyl-b-D– maltoside, pH ¼ 7.2 (T ¼ 293 K) See Table for abbreviations NF, no formation of QA was detected; ND, not determined; NP, no acceptable placement was found by FLEXX Quinone E1/2a (mV) kAD (s)1) Dr(NQ) ˚ (A) NQ 2MNQ 5MNQ 6MNQ 23DMNQ 25DMNQ 26DMNQ 27DMNQ 28DMNQ 58DMNQ 67DMNQ 235TMNQ 236TMNQ 258TMNQ 267TMNQ 2358TeMNQ 2367TeMNQ 5678TeMNQ 25678PMNQ HMNQ 2UNQ 2M3UNQ 25DM3UNQ 26DM3UNQ 27DM3UNQ 28DM3UNQ 258TM3UNQ 267TM3UNQ PM3UNQ )1057 )1146 )1150 )1091 )1227 )1218 )1173 )1179 )1224 )1228 )1132 )1297 )1279 )1300 )1209 )1386 )1280 )1305 )1391 )1487 )1099 )1206 )1288 )1261 )1255 )1295 )1366 )1294 )1477 7.1 6.2 10.2 10.6 6.6 9.4 8.8 14.4 8.4 NF 13.8 12.0 11.2 NF 15.0 NF 17.1 NF NF NF 9.3 8.8 5.7 14.3 17.7 16.8 NF 24.8 NF 0.00 )0.05 0.08 )0.31 )0.45 )0.38 )0.44 )1.05 )0.02 NF )0.49 )0.17 )0.64 NF )0.51 NF )0.69 NF NF NF 0.07 )0.08 )0.50 )0.76 )0.76 )0.05 NF )0.84 NF DG0 (kJỈmol)1) bind –log Kd DG0 app (kJỈmol)1) log Pcw log Pcwb Exp FLEXX Dd 5.4 6.1 5.6 6.3 8.1 6.0 7.2 7.1 6.6 )16.8 1.21 1.80 1.68 1.68 2.39 2.27 2.27 2.27 2.27 2.27 2.27 2.86 2.86 2.73 2.73 3.33 3.33 3.08 3.67 4.26 5.76 6.36 6.82 6.82 6.82 6.82 7.52 7.52 8.22 1.26 1.88 – – 2.70 – – – – – 2.17 – – – 2.72 – 3.48 – – – – – – – – – – – – )23.5 )24.1 )22.0 )25.9 )32.0 )21.0 )27.6 )27.1 )24.3 ND )26.6 )27.2 )31.1 ND )24.0 ND )25.1 ND ND ND )6.4 )13.1 6.2 )9.4 )5.5 )3.8 ND 0.1 ND )23.9 )25.8 )23.4 )23.4 )25.0 )22.3 )25.5 )22.7 )25.3 )16.0c )20.0 )23.9 )23.7 NP )22.0 NP )22.6 NP NP NP )22.1 )21.4 )14.9 )19.8 )16.6 )20.6 NP )17.6 NP )0.4 )1.7 )1.4 2.5 7.0 )1.3 2.1 4.4 )1.0 – 6.6 3.3 8.4 – 2.0 – 2.5 – – – )15.7 )8.3 )21.1 )10.4 )11.1 )16.8 – )17.7 – a In vitro midpoint redox potentials for the redox couple Q/Q– reported against ferrocene as internal standard measured in DMF [31,53] Experimental data taken from [54] c Only one hydrogen bond between the carbonyl oxygen of the quinone and surrounding amino acid residues was found d D  DG0 ðFLEXXÞ À DG0 ðExp:Þ bind bind b successfully docked into the binding pocket, except for of the compounds containing methyl groups in both positions and Quinone orientation within the binding pocket In all predicted structures the 1,4-naphthoquinones share essentially the same orientation of the naphthoquinone ring system, i.e the calculated protein–ligand complexes show two hydrogen bonds between the carbonyl oxygens of the quinones and the amide nitrogen of AlaM260 and the imidazole nitrogen of the HisM219 side chain as found for UQ-10 in the X-ray structure The position and the orientation of the quinone rings are similar to those of UQ-10 (Fig 3) The aromatic rings of the naphthoquinones are directed towards the interior of the binding pocket (i.e towards MetM262 and AlaM245) Up to eight specific interactions of the aromatic rings with side chain methyl groups (AlaM248, AlaM249, AlaM260, Cc2 of ThrM222 and IleM265), aromatic rings (TrpM252) and backbone amide bonds (AlaM248, AlaM260, ThrM261) of surrounding amino acids were assigned by FLEXX In case of the quinone oriented with two hydrogen bonds of the carbonyl oxygens and the aromatic ring directed towards the interior of the binding site, the ring system can assume two orientations They can be matched by a rotation 1102 O Hucke et al (Eur J Biochem 269) Fig QÀ =QA FTIR difference spectra of quinone-depleted reaction A centers from R sphaeroides reconstituted with UQ-10, vitamin K1 (Vit K1), 2,3,5-trimethyl-1,4-naphthoquinone (235TMNQ), 2,8dimethyl-3-undecyl-1,4-naphthoquinone (28DM3UNQ) and 2,3,6trimethyl-1,4-naphthoquinone (236TMNQ) A total of 40 000 interferograms were averaged for each spectrum As shown for vitamin K1 and UQ-10, the regions of 1750–1670 cm)1 and 1560–1500 cm)1 are free of quinonic contributions (indicated as nonquinonic) [41] Differences in the structure of the binding site due to structural differences of the primary quinone at QA are expected to alter the vibrational frequencies and intensities of the spectra in these regions See text for details of conditions a.u absorbance units of 180° on the x-axis (Fig 3) but are only distinguishable for asymmetrical methyl substitution patterns We arbitrarily chose the naphthoquinone orientation with the hydrogen bonds between the C1 carbonyl and HisM219 and the C4 carbonyl and AlaM260 as reference (designated reference orientation) In the calculated structures, for all naphthoquinones with this reference orientation the methyl groups at a specific position of the naphthoquinone ring system have very similar environments which we used for the definition of methyl group positions within the QA binding site (Table 3) Each position is defined by the contacts that are observed in the predicted structures between a specific methyl group and the RC atoms It is named according to the number of the C-atom of the naphthoquinone to which the methyl group is bound For instance, the ¢position 5¢ is formed by all RC atoms showing contacts to the 5-methyl group A % 180° rotation on the x-axis moves a methyl group from position 5, and to position 8, and 3, respectively The detailed analysis of the placements of the tailless naphthoquinones reveals that the presence of specific methyl substituents favors one of the two possible orientations with respect to the x-axis: In all placements with 5- or 8-substitution (5MNQ, 25DMNQ, 28DMNQ, 235TMNQ) Ó FEBS 2002 Fig Comparison of the ubiquinone-10 (UQ-10) position at QA in the photosynthetic reaction center from R sphaeroides obtained from docking calculations with the X-ray structure Carbon atoms of the UQ-10 from [38] and the docked quinone are depicted in black and green, respectively Amino acids (blue) surrounding UQ-10 were taken from the crystal structure ([38], PDB file 1AIJ) The nitrogen, oxygen and hydrogen atoms are drawn in light blue, red and white, respectively For sake of clarity the isoprenoid chains were truncated The dashed lines indicate the hydrogen bonds between the carbonyl oxygens of the quinones and AlaM260 and HisM219 The quinone head ˚ groups fit with an rmsd of 0.29 A the corresponding methyl group was found at position The best placements (with regard to the FLEXX score of the binding free energy) of these quinones with the methyl group at position in the binding pocket show binding free energies which deviate from the optimal value corresponding to placement by 3.9, 1.7, 6.9 and 3.5 kJỈmol)1, respectively In the best placements of quinones with 6- or 7-methyl substitution (6MNQ, 26DMNQ, 27DMNQ, 236TMNQ) the methyl group was found at position Compared to these placements structures with the methyl group at position of the binding site exhibit less favorable energies of 3.1, 6.4, 0.1 and 2.4 kJỈmol)1, respectively The favored placement of 2MNQ shows the methyl group at position and the corresponding binding free energy is 4.1 kJỈmol)1 lower than that of the best placement of this compound, rotated by approximately 180° on the x-axis (which places the 2-methyl group at position 3) This also accounts for the small energy differences of 1.7 and 0.1 kJỈmol)1 between the two rotated orientations of 25DMNQ and 27DMNQ, respectively Here, the placement of the 5-methyl group to position and the 7-methyl group to position leads to the unfavorable position of the 2-methyl group Evaluating the docked structures of the naphthoquinones with an undecyl tail we found for 2UNQ the rotated orientation of the quinone head group with hydrogen bonds between the C1-carbonyl group and AlaM260 and Ó FEBS 2002 Vitamin K derivatives at the QA-site (Eur J Biochem 269) 1103 Table Possible orientations of methylated 1,4-naphthoquinones in the QA binding site as determined with FLEXX The methyl groups of the different methylated 1,4-naphthoquinones occupy distinct positions within the QA binding site as defined by their contacts with amino acid atoms of the reaction center Each position number refers to the C atom number of the naphthoquinone ring (see Scheme 1) to which the corresponding methyl group is bound It was assumed that the naphthoquinone is located in the most common orientation with the two hydrogen bonds between the C1 carbonyl and the C4 carbonyl formed to HisM219 and AlaM260, respectively (arbitrarily defined as the Ôreference orientationÕ) Position within the QA site Fig Calculated position of 2,6-dimethyl-3-undecyl-1,4-naphthoquinone (26DM3UNQ) in the QA binding pocket as an example of the predicted structures with 1,4-naphthoquinones as primary acceptors of the RC from R sphaeroides The ring systems of both compounds show a high similarity in terms of the position and orientation x and y denote rotational axis used for the description of the quinone orientation within the QA binding site See Fig for coloring the C4-carbonyl group and HisM219 However, in all six 2methyl-3-undecyl-1,4-naphthoquinone derivatives the same quinone head group orientation was observed as in the reference orientation of the tailless quinones For these compounds the rotated reference orientation is prohibited as the presence of the 2-methyl group prevents a necessary adjustment of the undecyl tail conformation directing the hydrocarbon tail through the opening of the binding pocket towards the protein exterior For the same reason, in the predicted structure with 25DM3UNQ the 5-methyl group is not found at position of the binding site (as found for all 5-methylated tailless naphthoquinones, see above) but in the less favorable position It should be mentioned that for most of the tailless quinones placements with the aromatic rings directed towards the opening of the binding pocket were also computed This orientation is obtained by a rotation of % 180° on the y-axis relative to the reference orientation, designated Ôantireference orientationÕ) However, the binding free energies are significantly larger by an average of +5.4 kJỈmol)1 compared to the highest ranked placements indicating that this orientation is less favorable The analysis of the different free energy terms of the scoring function reveals that the main contribution (% 47%) of this increase in the binding free energy results from a loss of aromatic interactions Orientation of the aromatic naphthoquinone rings to the opening of the binding site reduces the average number of these interactions from 5.4 to 1.9 suggesting that these contacts play an important role for naphthoquinone binding to the QA binding site Amino acids Amino-acid atoms HisM219 TrpM252 IleM265 TrpM252 MetM256 AlaM249 AlaM260 AlaM245 AlaM249 AlaM260 ThrM261 MetM262 HOH 64, 409 AlaM248 MetM262 HisM219 IleM223 MetM262 IleM265 Ca, Cd1 Cd1, Ne1 Cd1, Cc1, Ca Cb, Cc, Cd1 Ce Ca, Cb, N C, Ca, N, O Cb, O Cb O C, Ca N O Cb Ca, Cb, Cc, Sd, Ce Ce1 Cd1 Sd, Ce Cc2 Other contributions to the change in the binding free energy include the loss of lipophilic contact area (% 15%) and deviations from the ideal hydrogen bond geometry (% 38%) Distances from the naphthoquinone at QA to the secondary ubiquinone QB and the primary donor D According to the Marcus theory the rate of electron transfer between two molecules depends on three factors: the overlap of the electron densities (wavefunctions) of the two molecules, the difference in redox potential of the molecules (corresponding to the free energy difference) and the reorganization energy (reviewed in [42]) The most critical parameter in determining the electron transfer rate is the electron density overlap which was found to depend exponentially on the distance of the reactants [43] The center-to-center distances are measured from the middle of the quinone rings and the center of the special pair (defined as the middle of the line connecting the Mg atoms) Due to different methyl substitution patterns the use of the edge-to-edge values produces incomparable values The distances between the different naphthoquinone compounds at QA and the native ubiquinone at QB ˚ ˚ ˚ range from 19.4 A to 20.0 A compared to 19.6 A as found in the X-ray structure of native RCs with ubiquinone at QA Thus, within the resolution of the X-ray diffraction data both structures are identical However, in terms of the Ó FEBS 2002 1104 O Hucke et al (Eur J Biochem 269) distance between the primary donor and QA some naph˚ thoquinones are further apart from the donor (up to 1.1 A in case of 5-methyl-1,4-naphthoquinone) than the native ubiquinone affecting significantly the electron transfer rate kAD (see Discussion) Evaluation of binding free energies The binding free energies of the protein-ligand complexes were estimated with the FLEXX scoring function (Eqn 7) For all functionally binding naphthoquinones without an undecyl tail the values range from )25.8 to )20.0 kJỈmol)1 The contributions of the hydrogen bonds, aromatic interactions and lipophilic contacts amount to % 32, % 13 and % 55%, respectively In case of 58DMNQ a protein-ligand complex was predicted although with the charge recombination assay no binding was observed However, the binding free energy yielded a significantly higher value of )16 kJỈmol)1 mainly due to the loss of one of the two hydrogen bonds This compound was therefore disregarded in all further analysis The calculated binding energies of the substituted undecyl naphthoquinone derivatives range from )22.1 to )14.9 kJỈmol)1 Based on the different terms in the scoring function we deduced that the major component is the lipophilic contact energy (% 67%) due to the large hydrophobic surface of the alkyl chain Smaller contributions arise from the hydrogen bonds (% 24%) and interactions between the aromatic rings of the naphthoquinones and the residues of the binding site (% 9%) A detailed analysis reveals that the binding free energies of the quinone head groups (methyl substituted ring systems without undecyl chain) lie only slightly (on average 1.3 kJỈmol)1) above the energies found for comparable naphthoquinones without an alkyl chain, showing that the presence of the undecyl tail has no significant effect on the interaction between the quinone head group and the protein binding site As described above, the methyl groups of all 5-substituted quinones except that of 25DM3UNQ were found at position within the binding pocket in the calculated complexes (Table 3) In these structures, the coordinates of the methyl group were practically identical leading to the same protein environment formed predominantly by the side chains of HisM219, IleM223, MetM262 and IleM265 (Table 3) We have constructed hypothetical protein-ligand complexes of the nonbinding compounds 58DMNQ, 258TMNQ, 2358TEMNQ and 258TM3UNQ based on the predicted structures with the corresponding binding analogues 5MNQ, 25DMNQ, 28DMNQ, 235TMNQ, 25DM3UNQ and 28DM3UNQ For this purpose, the ring systems of each of the 5,8-disubstituted naphthoquinones and the corresponding monosubstituted quinone in the computed protein-ligand complex were superimposed All resulting hypothetical structures share the same features (Fig 4) The additional methyl group shows an intolerable van der Waals overlap with either the backbone atoms of AlaM260 and AlaM249 in case of 58DMNQ, 258TMNQ, 2358TEMNQ whereas for 258DM3UNQ steric clashes with the side chains of HisM219, IleM223 and MetM262 are found This can not be avoided by a displacement of the quinone head group within the binding site, as the methyl group of the 5- or 8-monosubstituted quinone is already in close contact with the adjacent part of the binding pocket restricting the positional freedom of the quinone Naphthoquinone positions: implications for the charge recombination rates A main assumption for comparing the different naphthoquinone positions is that the original structure of the QA binding pocket remains unchanged in view of the drastic methods including the application of high concentrations of DISCUSSION Binding or nonbinding? The experimental values for the dissociation constants manifested that 5,8-disubstitution of the naphthoquinone system prohibits binding to the QA site even in case of a long tail in position This result was rather surprising as Warncke and Dutton found for 3-decyl substituted ubiquinone-0 and 2-methyl-1,4-naphthoquinone a decrease in the dissociation constants by more than two orders of magnitudes compared to the corresponding analogues with a hydrogen at this position [28] Our findings agree with previously published results obtained for 58DMNQ [44] As only functional binding is detected with the charge recombination assay it cannot be decided whether 5,8-dimethyl-1,4-naphthoquinone compounds were not bound at QA or the structure of the RC-quinone complex prohibits photoreduction However, the experimental data coincide strikingly with the results of the docking calculations: For none of these compounds an acceptable protein–ligand complex was found which strongly supports the idea of nonbinding to QA This can be pinned down to steric reasons: Fig Constructed placement of 5,8-dimethyl-1,4-naphthoquinone (58DMNQ) in the QA binding site compared to the native structure For reasons of clarity, the part of the binding site formed by IleM223 and MetM262 is drawn schematically as blue curve The pink dashed lines symbolize steric clashes of the 5-methyl group with (mainly backbone) atoms of AlaM260 and AlaM249 prohibiting binding of this quinone See Fig for color scheme Ó FEBS 2002 Vitamin K derivatives at the QA-site (Eur J Biochem 269) 1105 ionic detergent (LDAO) and inhibitor (o-phenanthroline) to remove the native ubiquinone from the binding site Breton – et al [45] measured the QA/QA FTIR difference spectra with native RCs having UQ-10 as QA and compared the result with that of quinone–depleted RCs after reconstitution of the QA-binding pocket with UQ-10 Within the noise level, the two spectra were practically identical More recently, Kuglstatter et al [46] determined the X-ray structure of the photosynthetic RC from R sphaeroides reconstituted with ˚ 9,10-anthraquinone as QA to 2.4 A resolution Quinonedepleted RCs were prepared under the same conditions as described in this work Within the resolution limit no structural changes of the QA binding pocket were observed From our predicted placements it follows that the position of 1,4-naphthoquinones within the QA binding pocket of the reaction center varies depending on the substitution pattern of the naphthoquinone This slightly affects the distances of QA to other cofactors involved in electron transfer reactions which may influence the rates of these reactions The differences are neglectable for the – forward electron transfer from QA to QB whereas the deviations are more critical with respect to the distance from the primary donor to QA According to the Marcus theory, at room temperature the charge recombination rate kAD depends on the reorganization energy, the standard reaction free energy and the electronic coupling matrix element (designated VR at the distance R between the reactants) To estimate the effect of quinone relocation in the calculated complexes on the rate kAD we use in a simple model the expression for the distance dependence of the electronic coupling matrix element (Eqn 8) by ignoring any possible changes in the reorganization energy, the driving force and the electron transfer pathway upon substitution V2 ẳ V2 expbRị R 8ị Here, V0 is the maximum electronic coupling matrix element, R is the distance between the reactants and b is the transmissional coefficient For this quantity Moser et al ˚ [43] have empirically determined a value of b ¼ 1.4 A)1 The maximum difference with respect to the donorquinone distance was found between 5MNQ and 27DMNQ ˚ (Table 2) The position of the latter compound was % 1.1 A closer to the donor leading to an approximate fivefold increase in the charge recombination rate kAD With respect to ubiquinone as found in the X-ray structure a mean ˚ displacement of % 0.6 A away from the donor was determined for the different naphthoquinone compounds This displacement may account for a % 2.4-fold decrease in the rate kAD Evidence for position-dependent influences on the rate kAD was previously reported by Warncke et al [28] They studied both menaquinone and ubiquinone compounds with systematically altered hydrocarbon tail structures Using the empirical relation for the distance dependence of the electron transfer rate in proteins of Moser et al [43] ˚ the relocation of the quinones was estimated to 0.8 A and ˚ along the line connecting the quinone and the primary 0.6 A donor, respectively Similar values were derived from FLEXX calculations on quinones with systematically altered hydrocarbon chain length (data not shown) Gunner et al [5] ˚ proposed positional differences of about A to explain a three to fourfold increased recombination rate for b- compared to a-substituted 9,10-anthraquinones Moreover, in the X-ray structure of the reaction center from R sphaeroides with 9,10-anthraquinone as QA [46] its ˚ position was found to be % A displaced compared to that of ubiquinone The docked anthraquinone-reaction center structure exhibits very similar results (data not shown) Comparison of experimental and calculated binding free energies 1,4-Naphthoquinones without undecyl chain According to our model, the free energies of the quinone transfer from the aqueous solution to the hydrophobic detergent and protein-detergent micelles were estimated by calculating the free energies for the transfer from water to cyclohexane The experimental binding free energies were corrected for these transfer energies to account for the tendency of the hydrophobic quinone compounds to accumulate within the hydrophobic micellar phase Although this correction is based on a relatively simple model, we achieved a reasonable agreement between theoretical and experimental binding free energy values (Table 2) The standard deviation of the two data sets amounts to only 3.9 kJỈmol)1 1,4-Naphthoquinones with undecyl chain The experimental binding energies of the naphthoquinones with a 3-undecyl chain display on average an offset of 14.4 kJỈmol)1 compared to the predicted values Warncke & Dutton [33] found empirically that the binding free energies (DG0 ) of bind many quinones to the QA site can be corrected with respect to their hydrophobic transfer free energies (DGtrans ) by applying a simple linear relationship yielding a measure for the direct interactions of the protein with the ligand In case of ubiquinones with more than two isoprene unit tails, corresponding to a linear chain length of eight carbon atoms, this correction method failed [28] This was explained with the third and subsequent isoprene units being not completely removed from contact with the solvent Therefore, DG0 is expected to be overestimated for naphthotrans quinones with a hydrocarbon chain of 11 carbon atoms with our method as well Other possible sources of error include inaccuracies of the lipophilic contact energy by the scoring function of FLEXX and of the calculated partition coefficients Pcw Under our experimental conditions the apolar phase was not cyclohexan but consists of both, detergent micelles and mixed micelles of detergent and protein Assuming that a systematic error in estimating the effective energy for the transfer of the undecyl-naphthoquinone compounds from water to the mixed protein-detergent micelles accounts for the discrepancies, a simple offset correction based on the average values of the experimental and predicted values matched the two data sets with a standard deviation of 4.1 kJỈmol)1 (data not shown) Aromatic interactions of the naphthoquinone compounds with the QA binding site From our calculations it follows that the interactions between the aromatic rings of the tailless naphthoquinone derivatives and the protein play an important role with respect to the quinone orientation within the QA binding site The binding free energy associated with these interactions Ó FEBS 2002 1106 O Hucke et al (Eur J Biochem 269) amounts to only % 13% of the total energy, representing the smallest of the different contributions (Eqn 7) In contrast to this finding, the aromatic interactions represent the major component with respect to the orientation of the quinones Approximately 46% of the energy difference between the best placements of the reference and the antireference orientation arises from these interactions A structural alignment of the reaction centers from B viridis and R sphaeroides shows that the positions of all groups of the QA binding site, involved in the aromatic interactions are highly conserved This indicates, that aromatic interactions may be important for the binding of the physiological primary electron acceptor of the reaction center from B viridis, vitamin K1, which is a naphthoquinone derivative The aromatic interactions of the naphthoquinone with the QA binding site in contrast to ubiquinone might be the reason for the failure of ubiquinone to replace naphthoquinone at this site This is the prerequisite for the preparation of RCs with naphthoquinone compounds at QA and ubiquinone at QB (Ôhybrid RCsÕ), enabling mechanistic studies of the forward electron transfer from the primary to the secondary quinone [18,19] and of the direct charge – recombination process from QB and D+ [16] Accuracy of the calculated binding free energies To estimate the binding free energies of the placements, we used the empirical scoring function introduced by Bohm ă [47], with minor modications This function was tested by evaluating the X-ray structures of 82 different proteinligand complexes displaying experimentally determined binding free energies in the range from )8 to )80 kJỈmol)1 These data were reproduced with a standard deviation of 9.5 kJỈmol)1 [48] However, a much better result was obtained in a test case which is similar to our docking study of naphthoquinone compounds: Five different, structurally closely related inhibitors of dihydrofolate reductase were docked by hand into the binding pocket of the enzyme by use of computer graphics [47] The scoring function was applied to the modeled protein-ligand complexes, yielding a standard deviation from the experimental binding energies of only 4.0 kJỈmol)1 This value obtained for a water soluble protein is nearly identical with the rmsd of the calculated and experimentally determined binding free energies of the different naphthoquinones to the QA site of the R sphaeroides RC indicating that our method to correct the apparent binding energies for the hydrophobic transfer energies works well (at least for the tailless naphthoquinones) In addition, the scoring function seems to be well suited for the characterization of the interactions between the naphthoquinones and the QA binding site To the best of our knowledge this is the first application of FLEXX to a membrane protein with a binding site being inaccessible from the aqueous phase The good agreement of the predicted with the experimentally observed naphthoquinone binding properties shows that docking calculations with FLEXX provide a powerful tool for the rational design of new artificial electron acceptors and inhibitors of other quinone binding proteins in the context of the recently determined structures of photosystem II [49], mitochondrial bc1 complex [50] and fumarate reductase [51,52] ACKNOWLEDGEMENTS We thank Ursula Friedrich for growing the bacterial cultures, isolating and purifying the reaction centers and Peter Graber for general support ¨ which made this work possible This work was supported by a grant from the Deutsche Forschungsgemeinschaft (La 816/3–3) REFERENCES Feher, G., Allen, J.P., Okamura, M.Y & Rees, D.C (1989) Structure and function of bacterial photosynthetic reaction centers Nature 339, 111–116 Blankenship, R.E., Madigan, M.T & Bauer, C.E (1995) Anoxygenic 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Photosynth Res 56, 75–82 Itoh, S & Iwaki, M (1989) Vitamin K1 (phylloquinone) restores the turnover of FeS centers in the ether-extracted... free energy, was considered for further analysis The binding site of the primary quinone in the reaction center protein from R sphaeroides was determined with the molecular modeling package WHATIF