Localization of Two Phylloquinones, QK and QK', in an Improved Electron Density Map of Photosystem I at 4-Å Resolution*

Olaf KlukasDagger , Wolf-Dieter SchubertDagger §, Patrick JordanDagger , Norbert KraußDagger , Petra Frommeparallel , Horst Tobias Wittparallel , and Wolfram SaengerDagger

From the Dagger  Institut für Kristallographie, Freie Universität Berlin, Takustraße 6, D-14195 Berlin, Germany and parallel  Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität, Straße-des-17, Juni 135, D-10623 Berlin, Germany

    ABSTRACT
Top
Abstract
Introduction
References

An improved electron density map of photosystem I from Synechococcus elongatus calculated at 4-Å resolution for the first time reveals a second phylloquinone molecule and thereby completes the set of cofactors constituting the electron transfer system of this iron-sulfur type photosynthetic reaction center: six chlorophyll a, two phylloquinones, and three Fe4S4 clusters. The location of the newly identified phylloquinone pair, the individual plane orientations of these molecules, and the resulting distances to other cofactors of the electron transfer system are discussed and compared with those determined by magnetic resonance techniques.

    INTRODUCTION
Top
Abstract
Introduction
References

The electron transfer processes of oxygenic photosynthesis, as observed in cyanobacteria, eukaryotic algae, and higher plants, involve two distinct types of photosynthetic reaction centers located in the thylakoid membrane. Photosystem II catalyzes the light-driven luminal oxidation of water and the reduction of plastoquinone near the stromal side of the photosynthetic membrane. Photosystem I (PSI)1 luminally oxidizes the soluble electron donor plastocyanin (alternatively cytochrome c6) and stromally reduces the extrinsic electron acceptor ferredoxin or flavodoxin. The reduced ferredoxin induces the reduction of NADP+, a reaction catalyzed by ferredoxin:NADP+ reductase. Photosystem I receives electrons from photosystem II via an intermediate plastoquinone pool, the cytochrome b6/f complex, and water soluble electron carriers. The difference in proton concentration across the thylakoid membrane, which results from the proton pumping of the plastoquinone pool and the cytochrome b6/f complex, the stromal consumption of protons by NADP+ reduction, and the luminal release of protons following water oxidation, is used by the ATP-synthase for phosphorylation of ADP to ATP (1, 2).

Cyanobacterial PSI consists of 11 subunits referred to as PsaA to PsaF and PsaI to PsaM. An x-ray structural model of a cyanobacterial PSI complex from the thermophile Synechococcus elongatus has been postulated on the basis of an electron density map calculated at 4-Å resolution (3, 4). Despite the comparatively low resolution, it was possible to suggest an assignment of 43 alpha -helices to the individual subunits of PSI by correlating the information provided by the electron density map with available biochemical and biophysical data. Furthermore, the electron density map allowed the positions of 89 Chl a molecules, constituents of both the core antenna system and electron transfer system, one phylloquinone, and three iron-sulfur clusters to be modeled.

The electron transfer reactions of PSI are initiated through excitation of the primary electron donor P700 positioned near the luminal side of the membrane-integral complex. Structurally, P700 consists of a chlorophyll a dimer (eC1/eC1'), whose mutually parallel dihydroporphyrin ring planes are aligned with the membrane normal. Upon excitation, P700* passes an electron to the primary electron acceptor A (probably eC2 or eC2'; see below). Spectroscopically, the first electron acceptor has been identified as A0, in all probability one (though possibly either) of the pair of Chl a monomers denoted eC3 and eC3' in the structural model of PSI (3, 4). This process occurs with a rate constant of about 5·1011 s-1 (5). The charge separation P700plusdu Abardot 0 is spatially extended across the membrane by electron transfer from the radical Abardot 0 to the next electron acceptor spectroscopically referred to as A1; the rate constant is estimated to be 2-5·1010 s-1 (for a review, see Ref. 5). A1 is now generally agreed to be a phylloquinone (6, 7). Due to the difficulty of locating the small phylloquinone molecules in low resolution electron density maps and because of the stability of the radical state P700plusdu Abardot 1, the position and orientation of A1 relative to the PSI holocomplex has recently received increased attention, especially by improved EPR techniques. These have, inter alia, determined the distance between Abardot 1 and P700plusdu to be ~25.4 Å (8, 9, 10). A relative position for A1 was derived through orientation-dependent pulsed EPR measurements on PSI single crystals (10). Geometrically, this position was found to correspond to QK, a single phylloquinone assigned to a well defined pocket in the earlier electron density map (4). The assignment of this position, however, remained internally uncorroborated, since an expected pseudosymmetrically positioned second phylloquinone could not be identified at the time.

The three terminal cofactors of the electron transfer system are iron-sulfur centers, FX being closest to P700, followed by F1 and F2 (we retain this nomenclature, for the present, to emphasize the remaining structural ambiguity in their assignment to the known cofactors FA and FB, although see Refs. 11-13 as well as Ref. 14 for recent results correlating FA with F1 and FB with F2).

In the following, we describe an improved model of the electron transfer system of PSI based on the present electron density map at 4-Å resolution (14). This map reveals the position of the second phylloquinone molecule and allows the spatial positioning of all 11 cofactors of the electron transfer system of PSI. The positions and orientations of individual cofactors are discussed and compared with structural information derived from spectroscopic data.

    EXPERIMENTAL PROCEDURES

Calculation of an Improved Electron Density Map-- The phases for the electron density map presented here were derived using essentially the same data described previously (4), although a new native data set with a resolution of 3.5 Å and an additional mercury derivative data set have been included (14). Using the program SHARP (15) instead of the earlier combination VECREF/MLPHARE (16, 17) and including a total of five heavy atom derivative data sets, it was possible, by incorporating new minor sites, to derive a significantly improved heavy atom model. The program SOLOMON (16) has been employed in the solvent flattening procedure. Due to the low diffraction quality of heavy atom derivative crystals, experimentally obtained phase information is still limited to a resolution of 4 Å. Since no additional phase information at higher resolution could be achieved by phase extension using density modification techniques, the electron density map was calculated at a resolution of 4 Å. It reveals more detailed information on the polypeptide chain folding than previous maps as well as the complete cofactor set of PSI. For the detailed procedure and statistics for the determination of this electron density map, see Ref. 14.

Model Building-- The previously reported model of the electron transfer system (3, 4) has been used as a basis for the present cofactor model. The Chl a head groups are visible as almost quadratically flat density pockets. The positions and orientations of the Chl a molecules are modeled by 4-fold symmetrical porphyrin moieties, since the present resolution does not permit their asymmetric features to be defined unambiguously. Similarly, the phylloquinone molecules are represented by their naphthalene moieties to interpret the corresponding elongated ellipsoidal electron density. Neither the phylloquinone side chains nor the oxygen atoms have been included in the model.

Chl a cofactors of the electron transfer system were placed into the electron density using the program O (18), and their positions were optimized using the real space refinement procedure as provided by this program.

Distances between Cofactors and Associated Errors-- For Chl a molecules center-to-center distances were calculated between the central Mg2+ ions, while for iron-sulfur clusters and phylloquinones the centroid of the cluster and naphthalene model, respectively, have been used. Edge-to-edge distances of cofactors important for the kinetics of electron transfer were determined between the outer atoms of the porphyrin, naphthalene, and iron-sulfur cluster models, respectively. For iron-sulfur clusters, edge-to-edge distances have been determined between the iron and sulfur atoms of the clusters, as modeled.

The estimated errors for center-to-center and edge-to-edge distances are on the order of ±1 and ± 2 Å, respectively, the latter reflecting the larger uncertainties in the orientations of the planar cofactors within their molecular planes.

    RESULTS

Two Symmetrically Arranged Density Pockets Assigned to the Positions of the Phylloquinones-- In our previous x-ray structural model of PSI (4), only a tentative positional description of a single phylloquinone was included, assigned to an electron density pocket located between FX and eC3. The lack of a second, pseudosymmetrically positioned phylloquinone, however, prevented an internal corroboration of this identification.

The new electron density map now reveals two such electron density structures symmetrically positioned on either side of the pseudo-2-fold rotation axis C2(AB) and located between eC3 and FX. These have been assigned to the phylloquinone electron acceptors QK and QK' (Fig. 1). The latter is equivalent to the position QK identified previously (4). Note that following our earlier convention of priming cofactors coordinated by primed alpha -helices, the position previously denoted QK will be renamed QK' (coordinated by alpha -helices m'-n'), while the new second phylloquinone will be referred to as QK (coordinated by m-n).


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Fig. 1.   Stereo view of the electron density structures interpreted as QK and QK'. They are largely separate from the surrounding electron density corresponding to alpha -helices, inter-helical loops and eC3, eC3'. This figure was produced using O (18).

QK (QK') is situated slightly luminally of and close to the N terminus of alpha -helix n (n') and immediately adjacent to the loop connecting alpha -helices m and n (m' and n'). The corresponding electron density is clearly separated from that of the neighboring alpha -helices (Fig. 1). Facing away from the loop m-n (m'-n'), each phylloquinone is additionally delimited by the long loop n-o (n'-o') connecting the C-terminal end of n (n') to the stromal end of o (o').

In addition to QK and QK', a significantly more symmetrical arrangement of alpha -helices and connecting loops on either side of the pseudo-2-fold axis C2(AB) is now apparent in it vicinity as compared with the previously published electron density map. Whereas the earlier model of the alpha -helix m almost passed through the position now assigned to QK, the stromal end of m now has a comparable inclination relative to the membrane normal as its pseudosymmetric partner m'. The loops m-n (m'-n') connecting alpha -helix m (m') to the "surface" alpha -helix n (n') are similar in both shape and length (Fig. 2).


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Fig. 2.   The core of the reaction center of PSI. a, view direction perpendicular to the C2(AB)-axis onto median plane of all cofactors of the electron transfer system. The palisade of alpha -helices surrounding the electron transfer system and the loops connecting these alpha -helices are rendered in black and gray. The luminal loop i-j could not unambiguously be located in the electron density map. The naphthalene backbone of the modeled phylloquinone orientation as well as all remaining cofactors of the electron transfer system are depicted in white. b, view direction from the stromal side onto the membrane plane. The pseudo-C2(AB)-axis passes through the center of the Fe4S4 cluster FX. For clarity, only the stromal loop region j-k coordinating the iron sulfur cluster FX is shown. This figure was produced using Setor (37).

The electron densities of both QK and QK' are elongatedly ellipsoidal (Fig. 1). As a result, the long molecular axis of the naphthoquinone moiety may be identified with some confidence. However, the plane orientation as well as the quinone oxygen atoms remain indeterminate. As a result, the phylloquinone molecules have been modeled by their naphthalene backbones only. These naphthalene models were placed into the electron density optimizing their positions and the orientations of their long molecular axes. The molecular plane of QK was then rotated around the long axis to align the molecular plane with the vector eC1-QK, to account for the observation that the carbonyl O-O-axis is approximately aligned with the vector P700plusdu -Abardot 1 (19). Since the electron spin density is primarily located on either eC1 or eC1' (20) (although which one remains to be clarified), the procedure was repeated to align the molecular plane of QK with the vector eC1'-QK. Similarly, two plane orientations were obtained for QK'.

The long molecular axes of both QK and QK' are observed to be inclined by 13 ±5° relative to the membrane plane (equivalent to the crystallographic a,b-plane). Projected onto the a,b-plane, the long molecular axis of QK (QK') describes an angle of 18° (60°) to the crystallographic a-axis. The axes of QK and QK' form an angle of 42° with each other.

The Iron-Sulfur Clusters-- The positions of the iron-sulfur clusters correspond to the highest electron density observed (21). FX was tentatively modeled by fitting a Fe4S4 cluster into the electron density. Contouring the electron density map at 11 S.D. above the mean density reveals a tetrahedrally distorted electron density structure associated with FX (Fig. 3). The most likely explanation is, that this tetrahedron is equivalent to the arrangement of the four iron atoms of the Fe4S4 cluster. Modeling a Fe4S4 cluster into this tetrahedral shape results in a good structural match, while the four sulfur atoms lie outside the contour, in agreement with their lower density of electrons. Interestingly, the derived orientation of FX upholds the 2-fold symmetry of C2(AB), a fact that had been assumed on grounds of symmetry yet had remained unsubstantiated. The observation that the gXX principal axis of the g tensor of reduced FX is oriented perpendicular to the thylakoid membrane (22) now favors one of two alternative assignments of g tensor axes to the distorted cubane structure of Fe4S4 clusters. According to EPR studies on Fe4S4 model compounds (23, 24), our structural model and the EPR results are in agreement with the assignment, where each of the three principal magnetic axes is normal to one of the mutually orthogonal faces of the distorted Fe4S4 cube (25).


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Fig. 3.   Stereoscopic depiction of the electron density of FX contoured at 1.2 sigma  and at 11 sigma  above mean density. The tetragonal shape of FX in the 11 sigma  contour reveals the absolute orientation of FX observed to comply with the pseudosymmetry of C2(AB) (arbitrarily oriented axis C2(AB) not shown). For F1 and F2, such tetrahedrally shaped density structures are not evident. This figure was produced using BobScript (38).

The orientations of F1 and F2 have been inferred from the 2Fe4S4 ferredoxin structure from Peptostreptococcus asaccharolyticus (26) used as a model for PsaC (27). They are in agreement with those derived independently by EPR experiments on PSI single crystals (28). For F1 and F2, such tetragonally shaped density structures as observed in the case of FX are not evident as the electron density "outside" the membrane-integral region is less well defined.

    DISCUSSION

Overall Cofactor Arrangement-- The electron transfer system of PSI constitutes the innermost cylindrical core of the larger, membrane-integral photosynthetic reaction center complex. A set of 10 alpha -helices, five from each of the two central subunits, PsaA and PsaB, tightly encloses the electron transfer system, separating it from the surrounding antenna system (4). The electron transfer system itself consists of two symmetrically arranged cofactor branches (Fig. 2). Now that all cofactors of the electron transfer system have been identified, this pseudosymmetry is seen to encompass the whole of the membrane-integral region, extending from the pair eC1/eC1' near the luminal side to QK/QK' near the stromal side (Fig. 2). The iron-sulfur cluster FX is located on the pseudo-2-fold axis C2(AB), completing the symmetrical arrangement at the stromal edge of the membrane-integral subunits. Merely the two stromal iron-sulfur clusters F1 and F2 (FA and FB), coordinated by the extrinsic subunit PsaC, do not adhere to this 2-fold symmetry.

In the direction parallel to the membrane normal, the membrane-integral cofactors divide the membrane into four sections of roughly comparable width, here denoted eC1-eC2, eC2-eC3, eC3-QK, and QK-FX. The height difference for eC1-eC2, eC2-eC3, eC3-QK, and QK-FX amount to 5.9, 8.6, 7.8, and 8.8 Å, respectively, while the total distance eC1-FX is 31.1 Å. This corresponds to fractional distances of 0.19, 0.28, 0.25, and 0.28, respectively (Fig. 4a).


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Fig. 4.   Schematic representations of the electron transfer system. a, cofactor distribution along the membrane normal showing distances in Å and fractional distances. The observed center-to-center distances (±1 Å) (b) and the edge-to-edge distances (±2 Å) (c) are indicated. Except for the interplane distance between eC1 and eC1' (3.6 Å) the values of the other edge-to-edge distances have been determined with an accuracy of 0.5 Å. d, the individual distances and angles between the cofactors eC1, eC1', QK, and QK' are shown. They correspond to the center of the phylloquinones and are independent of the phylloquinone plane orientations. Present distances and angles are largely in agreement with those published previously (4). Note the schematic nature of these diagrams; true distances are supplied but may not be measured directly.

Photovoltage measurements on oriented PSI thylakoid membranes estimated values of fractional dielectrically weighted transmembrane distances of 0.62 for P700-A0 (compare with eC1-eC3, 0.47), 0.16 for A0-A1 (compare with eC3-QK, 0.25), and 0.22 for A1-FX (compare with QK-FX, 0.28) (29, 30). These relative distances, especially for the pair P700-A0, do not correspond to the x-ray structural model distances eC1-eC3 as well as one might have expected. Because the distances A0-A1 (eC3-QK) and A1-FX (QK-FX) are comparable (29), matching our observations, the distance P700-A0 (eC1-eC3) has clearly been overestimated by the photovoltage measurements relative to the other distances. Possibly, the fast rate of charge separation results in a significant error for the distance eC1-eC3 (alternatively, the dielectric constant around P700 may differ substantially from that nearer the middle of the membrane), giving rise to the observed distortion.

Intercofactor Distances from X-ray Structure and Spectroscopic Studies-- Comparisons of structural and spectroscopic data have recently been published based on models of PSI derived at 4.5- and 4-Å resolution (4, 5). These studies, however, included none or, in the latter case, a single phylloquinone position designated QK (now renamed QK'). Here we will include the latest structural results and compare these to the available spectroscopic data.

The Moser-Dutton "ruler" (31) (an empirical first order relationship between electron transfer rates and shortest edge-to-edge distances of the cofactors involved) provides a simple tool to estimate the "optimal" electron transfer rates from structural data, the optimal electron transfer rate being achieved when the sum of the standard reaction free energy and reorganization energy is essentially zero (32). In Table I, the edge-to-edge distances and the optimal (i.e. fastest theoretically possible) electron transfer rates derived, using the above relationship, are listed.

                              
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Table I
The averaged edge-to-edge distances between the cofactor planes and the "optimal" electron transfer rates derived using the Moser-Dutton relationship, log kET = 15 - 0.6R - 3.1(Delta G° + lambda )2/lambda (31).

Chlorophyll a Cofactors-- The Chl a molecules, eC1, eC1', eC2, eC2', eC3, and eC3' constitute the luminal half of the electron transfer system. eC1 and eC1' have been identified as structural components of the spectroscopically identified primary electron donor P700; eC2 and eC2' are referred to as the accessory chlorophylls; while either or both of eC3 and eC3' have been assigned to the spectroscopically identified primary electron acceptor A0.

As noted (4, 5), the edge-to-edge distance between eC1 and eC3 of 13.3 Å is too long to be compatible with the electron transfer rate of 5·1011 s-1 (for a review, see Ref. 5) determined for the primary electron transfer step (see Table I, Fig. 4c). It therefore seems likely that neither eC3 nor eC3' (the spectroscopically identified electron acceptor A0), but one or both of eC2 and eC2', represent an additional intermediate (i.e. true primary electron acceptor) with a calculated optimal electron transfer rate from eC1 to eC2 of 4·1012 s-1 and eC2 to eC3 of 1·1012 s-1 (see Table I).

The Phylloquinone Electron Acceptors-- The average center-to-center distance between QK and the neighboring cofactor eC3 amounts to 8.7 Å. Combining data from pulsed EPR experiments on the position of the quinone cofactor (relative to P700 and the crystallographic a-axis) with the x-ray structural model of PSI yielded a comparable center-to-center distance estimate of 7.5 ± 2 Å for eC3-QK (10).

The center-to-center distance between QK and FX has similarly been estimated to be 14 ± 2 Å by EPR measurements (10), matching that of the x-ray structural model (QK/QK')-FX = 14.3 ± 1/14.1 ± 1 Å (Fig. 4b). Although the orientation of the molecular plane of QK about the naphthalene long axis could not be determined from the electron density map, we determined the edge-to-edge distances to the surrounding cofactors. Compared with the overall errors estimated for edge-to-edge distances (±2 Å), the distances observed for the two QK plane orientations modeled prove insignificant.

The averaged edge-to-edge distance eC3-QK of 4.8 ± 2 Å is somewhat shorter than the value of A0-A1 <=  7.8 Å estimated from pico-nanosecond laser spectroscopy (33) (Table I). The difference between these values is possibly due to the inequivalence of the reaction free energy and reorganization energy, causing the distance to be overestimated (33). The averaged edge-to-edge distance between QK and FX is 11.3 Å ± 2 Å, which agrees well with the 10.7 Å suggested previously (34) (Table I).

The discrepancy between the electron transfer rate derived from the edge-to-edge distance eC1-QK and those rates reported for the charge recombination reaction P700plusdu Abardot 1 right-arrow P700A1 proves to be slightly more problematic; the latter is roughly 4·103 s-1 (5) (Table I). According to the Moser-Dutton approximation (31), the optimal (i.e. fastest possible) transfer rate for a direct recombination through the distance eC1-QK (20.5 Å) would be in the range of 5.0·102 s-1. The reason for the observed rates being faster than that calculated from the corresponding edge-to-edge distance for this pair of cofactors is unclear, although an intermediate step in recombination could provide an explanation of this difference.

Correlation of A1 with QK or QK'-- The identification of two phylloquinones and their introduction to the x-ray structural model gives new impetus to the question of which one of QK or QK' corresponds to the spectroscopically identified cofactor A1. To analyze the current possibilities, we derived two orientational models for each phylloquinone (see "Results"). The orientation and position of each of these models (two for each QK and QK') are quantified by the parameters shown in Fig. 5. They are successively compared with the corresponding values derived mainly from EPR experiments on oriented PSI particles or PSI single crystals (Table II).


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Fig. 5.   Schematic illustration of the geometric parameters used to describe the orientation of the phylloquinone molecules QK and QK'. a, side view (i.e. parallel to the membrane plane). alpha , angle  (vector P700plusdu -Abardot 1; c-axis). In addition to the phylloquinone centroid, the centers of the individual naphthalene model rings are used as reference points (dotted lines), since the electron spin density of Abardot 1 is presumably located exclusively on the ring bearing the oxygen atoms. Since these have not been located, both rings are considered. b, oblique view. beta , angle  (phylloquinone O-O-axis; c-axis); gamma , angle  (phylloquinone C2-Cmethyl bond; c-axis) (both orientations directed toward the stromal or luminal side are considered; the smallest value is given); delta , angle  (phylloquinone molecular plane; a,b-plane); epsilon , orientation of the phylloquinone long molecular axis relative to the a,b-plane. c, top view onto the a,b-plane. µ*, angle  (phylloquinone long molecular axis; a-axis); eta *, angle  (phylloquinone long molecular axis; vector P700plusdu -Abardot 1);phi , angle  (phylloquinone O-O-axis; vector P700plusdu -Abardot 1); omega *, angle  (vector P700plusdu -Abardot 1; a-axis); *, angles in projection onto the a,b-plane.

                              
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Table II
Geometric parameters describing the position and orientation of the phylloquinone molecules QK and QK'

The distance between P700plusdu and Abardot 1 has been placed at 25.4 ± 0.3 Å (8, 9). The center-to-center distances of all four combinations (eC1-QK, eC1'-QK, eC1-QK', and eC1'-QK') are compatible with the EPR data (Table II, Fig. 4d).

alpha -- The inclination of P700plusdu -Abardot 1 relative to the membrane normal (c-axis) is 27 ± 5° (10, 35). The corresponding values for eC1-QK, eC1'-QK, eC1-QK', and eC1'-QK' are 23, 28, 31, and 29 ± 2°, respectively (Table II, Fig. 4d; see also alpha  in Fig. 5). A unique assignment of P700plusdu Abardot 1 is thus not obtained, although eC1'-QK and eC1'-QK' give the closest agreement.

omega -- The angle between the projection of P700plusdu -Abardot 1 onto the a,b-plane and the a-axis (omega  in Fig. 5c) is put at 0 ± 10° by EPR simulations (10). It should be noted that because of the inherent D3 symmetry of these EPR techniques, the above values do not only hold for the crystallographic a-axis but also for the equivalent directions described by the vectors b and (a + b). In the following, the term "a-axis" therefore includes the true crystallographic a-axis, as well as its repetitions at 60° intervals in the a,b-plane. The corresponding values for omega  derived from the x-ray structural model are 12° (eC1-QK), 10° (eC1'-QK), 21° (eC1-QK'), and 5° (eC1'-QK'). Here eC1'-QK' yields the best agreement, although eC1'-QK and to a lesser degree eC1-QK cannot (entirely) be excluded.

phi -- The axis defined by the phylloquinone oxygen atoms has been reported to be essentially parallel (±10°) to the vector P700plusdu -Abardot 1 (19). Although the x-ray models of QK (QK') were derived by aligning the phylloquinone plane with the vector eC1-QK or eC1'-QK (eC1-QK', eC1'-QK'), corresponding to P700plusdu -Abardot 1, this leaves a possible deviation between the O-O-axis and eC1-QK within the phylloquinone plane (phi  in Fig. 5c). This deviation is observed to be 15 and 14 ± 10° (15 and 1°), respectively, for the vectors listed above, if the center of the phylloquinone is taken to represent the center of spin density of Abardot 1. These values change by as much as 3° if the electron spin density is assumed to be centered on either one of the naphthoquinone model ring systems (Table II). Here eC1'-QK' clearly shows the best correspondence to the EPR data.

beta -- Because EPR results indicate the phylloquinone O-O-axis to be aligned with P700plusdu -Abardot 1 (see above), the angle between the O-O-axis and the c-axis (denoted beta  in Fig. 5 and Table II) is assumed to be equivalent in size to the angle alpha  ( 208 (P700plusdu -Abardot 1; c-axis), Fig. 5a), i.e. 27°. Because of the observed deviation between the modeled O-O-axis and eC1-QK in the x-ray model, however, beta  ( 208 (O-O-axis; c-axis)) deviates by as much as 10° from 27° in the case of eC1'-QK (17°) and eC1-QK' (35°), although the agreement is quite good for both eC1-QK (27°) and eC1'-QK' (28°).

delta -- The phylloquinone plane of Abardot 1 is reported to be inclined by 76 ± 10° (35) relative to the a,b-plane (delta  in Fig. 5, Table II). For all x-ray structurally derived models, the inclinations are in the range 50-60°; the match between EPR- and x-ray structural values, therefore, is less than satisfactory.

gamma -- In the EPR model of Kamlowski et al. (36), the methyl group of the phylloquinones either faces the lumen or the stroma, while the C2-Cmethyl bonds subtend an angle of 35 ± 20° with respect to the c-axis (36). The equivalent value for the x-ray structural model phylloquinone plane orientations (gamma  in Fig. 5b, Table II) are in the range of 45-53°. Since the error of this value may be as large as 10°, the values do not necessarily contradict the EPR estimate of 35 ± 20° (36). This criterion, however, does not aid in distinguishing the four cofactor pairs of the x-ray structural model.

eta -- In the present x-ray structural model, the angles between the longest molecular axis of QK (QK') and the plane defined by the c-axis and the vectors equivalent to P700plusdu -Abardot 1 (eC1-QK, eC1'-QK, eC1-QK', and eC1'-QK') are 30, 68, 81, or 68°, respectively (eta  in Fig. 5, Table II). At present, no estimate for the angle eta  is available from spectroscopic investigations.

Combining the conclusions from the geometric comparison of the spectroscopic data and x-ray structural model indicates that eC1'-QK' shows the closest correspondence with the pair P700plusdu -Abardot 1. Nevertheless, the pairs eC1-QK and eC1'-QK similarly mostly remain within the error limits. Overall, eC1-QK' is most poorly correlated with the EPR data.

    CONCLUSION

A complete and internally consistent set of cofactors of the electron transfer system of PSI has been modeled into an improved electron density map at 4-Å resolution. The corrected model of the alpha -helices in the vicinity of the phylloquinone molecules leads to a better 2-fold symmetrical arrangement in this region of the PSI core. The distances for QK and QK' to other cofactors compare favorably with those suggested from spectroscopic measurements. Although the orientation of the phylloquinones remains undetermined about the long axis of the molecular plane, the pair P700plusdu -Abardot 1 is best correlated with eC1'-QK' followed by eC1-QK and eC1'-QK.

    ACKNOWLEDGEMENTS

We thank Andreas Kamlowski and Dietmar Stehlik (FU Berlin) for discussions and sharing data prior to publication. Synchrotron beam time provided by the EMBL outstation at ESRF (Grenoble, beamline ID2), LURE (Orsay, beamline DW32), SRS (Daresbury, beamline 9.6), EMBL outstation at DESY (Hamburg, beamline BW7B), and MPG-ASMB at DESY (Hamburg, beamline BW6) is gratefully acknowledged. Crystallization experiments under microgravity were made possible by the European Space Research and Technology Center of the European Space Agency during the Second United States Microgravity Laboratory and the Life and Microgravity Spacelab Missions of the National Aeronautics and Space Administration.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 312), the Bundesministerium für Bildung und Forschung, the Fonds der Chemischen Industrie, and the Deutsche Agentur für Raumfahrt-Angelegenheiten.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany.

To whom correspondence should be addressed. Tel.: 49 30 838 6326; Fax: 49 30 838 6702; E-mail: nkrauss{at}chemie.fu-berlin.de.

    ABBREVIATIONS

The abbreviations used are: PSI, photosystem I; PsaA and PsaB, large, central subunits of PSI, encoded by genes psaA and psaB; angle (a, b), angle between vectors a and b; a, b-plane, crystallographic plane parallel to the membrane plane; C2(AB), axis of pseudo-2-fold symmetry relating subunits PsaA and PsaB and also respective branches of the electron transfer system; Chl a, chlorophyll a; c-axis, crystallographic c-axis parallel to the membrane normal; eC1 and eC1', luminal Chl a cofactors of the electron transfer system and its pseudosymmetric counterpart; eC1, pertaining to both eC1 and eC1'; eC2 and eC3, second and third pair of Chl a cofactors of the electron transfer system; eCX, eCY, distances between named cofactor pairs (averaged value of pseudosymmetric branches); QK and QK', phylloquinone cofactors of the electron transfer system; F1 and F2, preliminary x-ray structural model names for FA and FB (FB and FA); P700, A0, A1, FX, FA, and FB, spectroscopically identified cofactors of the electron transfer system of PSI as follows: primary electron donor (dimer of Chl a molecules), primary (single Chl a), secondary (phylloquinone), intermediate, and two terminal (Fe4S4 clusters) electron acceptors; m, n, and o and m', n', and o', alpha -helix nomenclature.

    REFERENCES
Top
Abstract
Introduction
References
  1. Nugent, J. H. A. (1996) Eur. J. Biochem. 237, 519-531[Abstract]
  2. Witt, H. T. (1996) Ber. Bunsen-Ges. Phys. Chem. 100, 1923-1942
  3. Krauß, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H. T., and Saenger, W. (1996) Nat. Struct. Biol. 3, 965-973[Medline] [Order article via Infotrieve]
  4. Schubert, W.-D., Klukas, O., Krauß, N., Saenger, W., Fromme, P., and Witt, H. T. (1997) J. Mol. Biol. 272, 741-769[CrossRef][Medline] [Order article via Infotrieve]
  5. Brettel, K. (1997) Biophys. Biochim. Acta 1318, 322-373
  6. Sétif, P., and Bottin, H. (1989) Biochemistry 28, 2689-2697
  7. Snyder, S. W., Rustandi, R., Biggins, J., Norris, J. R., and Thurnauer, M. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9895-9896[Abstract]
  8. Dzuba, S. A., Hara, H., Kawamori, A., Iwaki, M., Itoh, S., and Tsvetkow, Y. D. (1997) Phys. Chem. Lett. 264, 238-244[CrossRef]
  9. Zech, S. G., Lubitz, W., and Bittl, R. (1996) Ber. Bunsen-Ges. Phys. Chem. 100, 2041-2044
  10. Bittl, R., Zech, S. G., Fromme, P., Witt, H. T., and Lubitz, W. (1997) Biochemistry 36, 12001-12004[CrossRef][Medline] [Order article via Infotrieve]
  11. Vassiliev, I. R., Jung, Y.-S., Yang, F., and Golbeck, J. H. (1998) Biophys. J. 74, 2029-2035[Abstract/Free Full Text]
  12. Díaz-Quintana, A., Leibl, W., Bottin, H., and Sétif, P. (1998) Biochemistry 37, 3429-3439[CrossRef][Medline] [Order article via Infotrieve]
  13. Fischer, N., Hippler, M., Sétif, P., Jacquot, J.-P., and Rochaix, J.-D. (1998) EMBO J. 17, 849-858[Abstract/Free Full Text]
  14. Klukas, O., Schubert, W.-D., Jordan, P., Krauß, N., Fromme, P., Witt, H. T., and Saenger, W. (1999) J. Biol. Chem. 274, 7351-7360[Abstract/Free Full Text]
  15. de La Fortelle, E., and Bricogne, G. (1997) Methods Enzymol. 276, 472-494
  16. Collaborative Computing Project 4. (1994) Acta Crystallogr. Sec. D 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
  17. Otwinowski, Z. (1991) in Proceedings of the CCP4 Study Weekend: Isomorphous Replacement and Anomalous Scattering (Wolf, W., Evans, P. R., and Leslie, A. G. W., eds), pp. 80-86, SERC Daresbury Laboratory, Warrington, United Kingdom
  18. Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sec. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
  19. van der Est, A., Prisner, T., Bittl, R., Fromme, P., Lubitz, W., Möbius, K., and Stehlik, D. (1997) J. Phys. Chem. B 101, 1437-1443[CrossRef]
  20. Käß, H., and Lubitz, W. (1996) Phys. Lett. 251, 193-203[CrossRef]
  21. Krauß, N., Hinrichs, W., Witt, I., Fromme, P., Pritzkow, W., Dauter, Z., Betzel, C., Wilson, K. S., Witt, H. T., and Saenger, W. (1993) Nature 361, 326-331[CrossRef]
  22. Guigliarelli, B., Guillaussier, J., More, C., Sétif, P., Bottin, H., and Bertrand, P. (1993) J. Biol. Chem. 268, 900-908[Abstract/Free Full Text]
  23. Rius, G., and Lamotte, B. (1989) J. Am. Chem. Soc. 111, 2464-2469
  24. Gloux, J., Gloux, P., Lamotte, B., Mouesca, J.-M., and Rius, G. (1994) J. Am. Chem. Soc. 116, 1953-1961
  25. Kamlowski, A., van der Est, A., Fromme, P., and Stehlik, D. (1997) Biochim. Biophys. Acta 1319, 185-198
  26. Adman, E. T., Sieker, L. C., and Jensen, L. H. (1976) J. Biol. Chem. 251, 3801-3806[Abstract]
  27. Schubert, W.-D., Klukas, O., Krauß, N., Saenger, W., Fromme, P., and Witt, H. T. (1995) in Photosynthesis: From Light to Biosphere: Proceedings of the 10th International Conference on Photosynthesis, Montpellier, August 20-25, 1995 (Mathis, P., ed), Vol. II, pp. 3-11, Kluwer Academic, Dordrecht, The Netherlands
  28. Kamlowski, A., van der Est, A., Fromme, P., Krauß, N., Schubert, W.-D., Klukas, O., and Stehlik, D. (1997) Biochim. Biophys. Acta 1319, 199-213[Medline] [Order article via Infotrieve]
  29. Leibl, W., Toupance, B., and Breton, J. (1995) Biochemistry 34, 10237-10244[Medline] [Order article via Infotrieve]
  30. Hecks, B., Wulf, K., Breton, J., Leibl, W., and Trissl, H.-W. (1994) Biochemistry 33, 8619-8624[Medline] [Order article via Infotrieve]
  31. Moser, C. C., Keske, J. M., Warncke, K., Farid, R. S., and Dutton, L. (1992) Nature 355, 796-802[CrossRef][Medline] [Order article via Infotrieve]
  32. Marcus, R. A., and Suttin, N. (1985) Biochim. Biophys. Acta 811, 265-322
  33. Iwaki, M., Kumazaki, S., Yoshihar, K., Erabi, T., and Itoh, S. (1996) Phys. Chem. 100, 10802-10809[CrossRef]
  34. Sétif, P., and Brettel, K. (1993) Biochemistry 32, 7846-7854[Medline] [Order article via Infotrieve]
  35. MacMillan, F., Hanely, J., van der Weerd, L., Knüpling, M., Un, S., and Rutherford, A. W. (1997) Biochemistry 36, 9297-9303[CrossRef][Medline] [Order article via Infotrieve]
  36. Kamlowski, A., Zech, S. G., Fromme, P., Bittl, R., Lubitz, W., Witt, H. T., and Stehlik, D. (1998) J. Phys. Chem. B 102, 8266-8277[CrossRef]
  37. Evans, S. V. (1993) J. Mol. Graphics 11, 134-138[CrossRef][Medline] [Order article via Infotrieve]
  38. Esnouf, R. M. (1997) J. Mol. Graphics 15, 132-134[CrossRef]


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