The Quinone Acceptor A1 in Photosystem I - American Chemical Society

The nature of the binding site of the quinone acceptor A1 in Photosystem I (PSI) is studied by modeling the protein and cofactor on the basis of struc...
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J. Phys. Chem. B 1998, 102, 8278-8287

The Quinone Acceptor A1 in Photosystem I: Binding Site, and Comparison to QA in Purple Bacteria Reaction Centers Andreas Kamlowski,†,‡ Brigitte Altenberg-Greulich,§ Arthur van der Est,*,⊥ Stephan G. Zech,⊥ Robert Bittl,⊥ Petra Fromme,⊥ Wolfgang Lubitz,⊥ and Dietmar Stehlik*,† Institut fu¨ r Experimentalphysik, Freie UniVersita¨ t Berlin, Arnimallee 14, 14195 Berlin, Germany; European Molecular Biology Laboratory, Abt. Biocomputing, Meyerhofstr. 1, 69117 Heidelberg, Germany; and Max-Volmer-Institut fu¨ r Biophysikalische Chemie und Biochemie, Technische UniVersita¨ t Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany ReceiVed: June 3, 1998; In Final Form: August 11, 1998

The nature of the binding site of the quinone acceptor A1 in Photosystem I (PSI) is studied by modeling the protein and cofactor on the basis of structural data derived from the intermediate resolution 4 Å X-ray diffraction electron density map, the position and orientation of A1 as evaluated from EPR data, and the histidine ligation of P700 as deduced from mutation experiments. Several models are constructed within the degrees of freedom allowed by the experimental constraints. In all cases a close interaction between the A1 headgroup and the side chain of PsaA-Trp697 (PsaB-Trp677) is found. The model is compared to the known binding site of QA in bacterial reaction centers (bRC) in which a similar quinone-tryptophan arrangement has been established. The results are also compared for consistency with published magnetic resonance data. The influences of the protein environment on the semiquinone g-tensor and hyperfine couplings are considerably different in PSI and bRC. It is argued that this is mainly a result of differences in the hydrogen bonding to the protein, in the strength of the π-π interactions with the tryptophan, and in the protein induced asymmetry in the spin density of the respective quinone radical anion.

1. Introduction High-resolution X-ray structures exist for some purple bacterial reaction centers (bRC)1a (see e.g. refs 1b-3). The intermediate resolution structure of Photosystem I (PSI) of a cyanobacterium4-6 represents the only structural characterization of another type of reaction center, in particular of plantlike photosynthesis. For this reason, efforts to correlate structural and functional properties in reaction centers have generally focused on bRC and, only recently, to some extent on PSI. It is generally accepted that reaction centers (RC) can be grouped into two classes and, conveniently, PSI and bRC are representatives of these two principal types. Of the many possible distinguishing features, the composition of the electron acceptor chains has been chosen for the classification of the two groups of RC’s into FeS-type (or type I) and Pheo-Q-type (type II), e.g. see refs 7, 8, and 18. The most prominent other representative of type II is Photosystem II (PSII) of oxygenic photosynthesis, whereas heliobacteria and green sulfur bacteria both have type I RC’s. In the two types of RC, light-induced electron transfer leads to stabilized charge separation across the membrane on a time scale of ∼10-10 s. In both cases this process occurs from the excited singlet state, 1P*, of the primary donor (P700 in PSI and P865 or P960 in bRC) via intermediate acceptors to the first quinone acceptor Q (A1 or QK in PSI and QA in bRC). In * Author to whom correspondence should be addressed. E-mail: [email protected]. Fax: +49 30-838/6081. † Freie Universita ¨ t Berlin. ‡ Present address: Institut fu ¨ r Organische Chemie, J. W. Goethe Universita¨t Frankfurt am Main. § EMBL Heidelberg. ⊥ Technische Universita ¨ t Berlin.

contrast, the subsequent electron transfer steps in the two types of RC differ strongly in terms of the nature of the terminal acceptors, rate and direction. In type II RC, the electron acceptor following QA is the secondary quinone QB, the transfer is comparatively slow (τ ∼ 100 µs) and the direction is primarily in the membrane plane. In type I RC, the acceptors are a series of three iron-sulfur (4Fe-4S) centers, the electron-transfer rate is orders of magnitude faster than in type II RC’s (τ ∼ 200 ns or faster) and the direction is mainly perpendicular to the membrane. Such major functional differences between the two types of RC are likely to be reflected in the properties of the respective primary quinone acceptor site. Indeed, the redox potential, solvent extractability, and EPR properties of the first quinone acceptor in the two types of RC are known to be very different. One of the prerequisites for a proper understanding of these functional differences is a knowledge of the structures of the two quinone binding sites at an atomic level. At present, this information is not available for PSI. On the other hand, many structural differences can be inferred from magnetic interaction parameters (g-tensors, hyperfine couplings (hfc), spin dynamics etc.). Many of these have been determined for both types of RC’s using a variety of magnetic resonance methods. Most of these data has been obtained from “trapped” paramag•netic states of the cofactors of interest (P•+ 700, A1 and reduced FeS centers). However, functional states, in particular •P•+ 700A1 , can also be studied directly using time-resolved EPR techniques. In the preceding paper9 we have obtained the orientation of the A1 quinone acceptor in the PSI reaction center from singlecrystal studies using transient EPR spectroscopy, consistent with the results obtained in ref 10. Combined with independent data on the location of the center of A1, this yields a nearly complete

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Quinone Acceptor A1 in Photosystem I structural model of the A1 headgroup, i.e., position and orientation within the overall cofactor arrangement as obtained from the intermediate resolution X-ray structure.6 In this paper, the focus is on the interaction of A1 with the protein environment with the aim of suggesting essential structural elements of the A1 binding site. Computer models of the likely key features of the A1 binding site in PSI are constructed using as input the position and orientation of A1, the orientation of the helical regions in the A1 environment as obtained from intermediate resolution X-ray data, the known primary sequences of the RC constituting proteins PsaA and PsaB, and relevant details of the QA binding site in bRC. The modeling results are then crosschecked by comparison with related properties of the quinone binding site in bRC. In particular, the wide range of magnetic •resonance data for A•1 and QA can be correlated to the known structure of the bRC binding site and the predicted structure of PSI. Since these data are of comparable extent and detail but only the structure of bRC is available at atomic resolution, it seems feasible to reach further conclusions about the A1 binding site on basis of just the EPR data. At this stage as the primary goal we will identify the structural similarities and differences among the functional quinone acceptor sites in the two RC types. In addition, this paper will address some aspects of the functional differences of the respective quinone sites and how they are related to structural differences. 2. Input Data for Modeling of the A1 Binding Site The model is based on a large series of different constraints from both experimental and theoretical origins. The experimental data does not contain enough information to uniquely build a model for the A1 binding site and consequently must be augmented with computationally derived data. We will briefly summarize and comment on the experimental data entering the model before presenting the results. 2.1. A1 Position and Orientation. The location and orientation of the phylloquinone headgroup of A1 in the active •state P•+ 700A1 have been determined with respect to the general cofactor arrangement of PSI from pulsed and transient, direct detection EPR on PSI single crystals.9,11 This information is summarized in Figure 8 of the preceding paper.9 The A1 position and orientation are shown with respect to the overall cofactor arrangement which includes the protein environment to the extent that it can be elucidated from the intermediate resolution electron density map.6 The experimental data on which this structural model of A1 is based are presented in detail in our previous paper9 along with a discussion of the assumptions and error limits. However, for the present context, a number of precautions should be mentioned once more that must be taken into account when using this information. It is most important to note that reliable information is currently lacking which identifies the active branch in the quasiC2 symmetric cofactor (and protein) arrangement. Similarly, it is not established confidently to which branch the main reaction center proteins PsaA and PsaB are associated. However, a choice has to be made to present the results as a figure. Accordingly, Figure 8 of the preceding paper gives only one of several possible representations consistent with the data. Both the functional A1 site (from EPR data) and the ground-state site QK suggested from X-ray data6 have been positioned into the same (left) cofactor branch, which corresponds to the one determined to be functional in bRC (see also Figure 1 below). Again, the choices made for Figure 8 of ref 9 are not based on experimental information, but they are necessary, in particular

J. Phys. Chem. B, Vol. 102, No. 42, 1998 8279 to visualize the comparison to the unambiguous arrangement in bRC. On the other hand, in the absence of definite experimental data for a distinction between the remaining choices, the analogy to bRC as chosen for the figures in this paper is the most plausible one. In this context, very recent experiments with deletion mutants concerning peripheral protein subunits12 show that removal of the specific subunits PsaE, PsaF, and PsaL changes the characteristics of the photoaccumlated A•1 EPR signal. Since these subunits are all distal to the C3symmetry axis of the PSI trimer (see e.g., ref 6), the results suggest the existence of just one active branch associated with the unprimed helices. Note that this is opposite to the choice made for the A1 (QK) site in Figure 8 of ref 9, and Figure 1 (see below). These new experimental data clearly underline the fact that the assignments made for the figures in this paper are preliminary. Alternative, consistent choices for the site allocations are equally valid. At present, structural constraints for the orientation of the A1 naphthoquinone headgroup are only published from EPR data.9,10 The location of the molecular center of the A1 headgroup has been determined both from the electron spin density center derived from pulsed EPR data of the functionally active A1 anion11 and from the electron charge density center of one of the two possible quinone acceptor sites obtained from X-ray data.6 2.2. Secondary Protein Structure and Sequence Analysis. For the modeling study, all structural information about the protein environment has been taken from the intermediate resolution electron density map.6 This includes coordinates for the axes of the helices m, n, and o (and their C2 symmetry related counterparts m′, n′, and o′) in the reaction center proteins PsaA/ B, the coordinates of the central Mg atoms of the pair of Chla molecules of P700 and of the center of the iron sulfur cluster FX. The latter coordinates provide appropriate and sufficient reference points to the overall reaction center structure. Scheme 1 shows the results of the secondary structure prediction and the transmembrane helix prediction in the area around the helices m, n, and o in PsaA and PsaB using the PHD prediction server.13-15 [Profile-based neural network prediction of secondary structure (PHDsec) and transmembrane helices (PHDhtm).] PHD predicts eleven transmembrane helices for each of the protein subunits, in agreement with the X-ray data.6 The PHD server not only provides a prediction but also indicates the reliability of the prediction by numbers on a scale from 0 to 9. In practice, if the reliability is 9, one can be 92% sure that the prediction is correct. Scheme 1 shows that the helices m, n, and o are all predicted by PHD with the highest possible reliability. Row 5 in Scheme 1, A and B, indicates the PHD transmembrane helix prediction result with the reliability factor underneath. Row 4 gives the same information for the overall prediction of helices. As expected, the n helix is strongly predicted to be helical, but is not predicted to be a transmembrane helix. Indeed, the X-ray structure6 indicates that the n helix is parallel to the plane of the membrane at the stromal surface. Finally, row 6 gives the transmembrane helical extensions suggested from hydrophobicity data analysis16 and the dots in bottom row 7 indicate the most reliable information about the extent of helix m from the electron density map.16 The alignment to the sequence will be inferred for helix m from the His ligation to P700 (see next subsection). Scheme 2 shows the sequence alignment of the regions containing the helices m, n, and o in PsaA and PsaB. A strong sequence homology (up to 55% sequence identity) exists. However, outside the transmembrane helices, only two regions

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SCHEME 1: Comparison of the Part of the Primary Amino Acid Sequences of Subunits PsaA and PsaB Used in Modeling the A1 Binding Site from Different Speciesa

a In both parts A and B, the first three rows show the part of the sequences of PsaA and PsaB from Synechococcus elongatus,16 Chlamydomonas reinhardtii,47 and Spinacia oleracea,48 respectively. The homology between the photosystems in cyanobacteria and plants is nearly complete. Homology between PsaA and PsaB is also strong (about 45% and somewhat higher with conservative replacements, see Scheme 2). The numbering corresponds to the sequence of the subunits from Synechococcus elongatus. Rows 4 and 5 give predictions of helical secondary structures based on helix prediction algorithms.14,15 Row 4: General helical motif. Row 5: Transmembrane helical motif. Underneath the H for helix, a reliability factor is given, the range of which is 0...9; the higher the factor, the better the prediction. The last two rows indicate corresponding regions of transmembrane helices inferred from experimental data. Row 6: Hydrophobicity plots.16 Row 7: Dots indicate the extension of helix m (m′) as inferred from the electron density map.6 The approximate locations of helices m(m′), n(n′), and o(o′) relative to the primary sequences are given in the last row (compare to Scheme 2).

SCHEME 2: Alignment of the Relevant Sections of the PsaA and PsaB Primary Sequences (cf. Scheme 1)a

a Conserved amino acids are related to each other by a solid bars and conservative replacements by dashed bars. The framed sections are regions of high homology outside the transmembrane helices. (Adopted and expanded from ref 17.)

of extended conservation exist: the FX binding region in the loop connecting helices j and k (not shown) and the marked regions in Scheme 2 between helix m and o. One of the framed regions in Scheme 2 extends from the end of helix m throughout most of helix n. Normally, a high degree of sequence conservation indicates a functional role. In this case, the functional role has been suggested6,17 to be quinone binding. Just before the stromal end of helix o, another highly conserved region is noted which also comes close enough to the presumed A1 location to be involved in quinone binding.18,19 However, there are no obvious candidates in this part of the amino acid sequence for quinone binding according to any of the common interaction mechanisms.

2.3. Histidine Ligation to the Mg Atoms of P700 and Sequence Alignment of Helix m. The histidine at position 680 in PsaA (660 in PsaB) is highly conserved. This suggests an important functional role for this residue. Several lines of evidence indicate that this histidine is a ligand to the central magnesium in one of the Chl a molecules of P700. First, this ligation of P700 is analogous to that of P865 in bRC (although His ligation is not the only possibility found in nature, see for example refs 20 and 21). Second, in the transmembrane helices m and m′ which pass closest to P700,6,22 PsaA-His680, and PsaB-His660 are the only histidines present. Third, sitedirected mutagenesis23-25 and isotopic labeling techniques26 also indicate that these two histidines bind P700. Using a His tolerant

Quinone Acceptor A1 in Photosystem I mutant from Synechocystis PCC 6803 together with ENDOR and ESEEM, Babcock and co-workers26 found evidence that a hyperfine coupling (hfc) exists between the electron spin on P•+ 700 and a nitrogen nuclear spin of a His residue. Simulation of the ESEEM data26 suggests that this coupling is primarily isotropic, indicating that the His is directly coordinated to the 27 Mg2+ ion of the spin-density carrying Chl a molecule of P•+ 700. In the most recent study,25 all conserved His residues of the so-called “reaction center domain” (with 10 transmembrane helices i (i′) to o (o′); cf. ref 6) of PSI from Chlamydomonas reinhardtii have been mutated to Glu or Leu. The properties of P700 (P•+ 700) (characterized using a combination of optical and FTIR difference spectroscopy and CW-EPR on P•+ 700) were affected only if the corresponding histidines, PsaA-His676 and PsaB-His656, were mutated. Redding et al.25 concluded that these His amino acids form the axial ligands of the two Chl a entities of P700. The His ligation to P700 is taken as the essential reference point to align the sequence and to orient and position helix m in the reaction center structure for the modeling. From the electron density map, helix m ends 12 residues beyond the His 680 (PsaA) which is also in line with the transmembrane helix prediction and hydrophobicity indication. Generally, the latter tends to give somewhat shorter helical ranges. 2.4. Location of the A1 Binding Site and Comparison with bRC. Taking all of the information summarized in the preceding sections, it is possible to locate the A1 binding site in the reaction center and to compare it with the QA binding site in bRC. The comparison of the two RC, restricted to the parts involved in P and Q binding, is shown in Figure 1. The RC structure of Rps. Viridis1 has been used for Figure 1 because QA is menaquinone which, like phylloquinone in PSI, has a naphthoquinone (NQ) headgroup. The position QK refers to the preliminary quinone ground state position obtained from an analysis of the electron density map at 4 Å resolution6 which 9,11 The is consistent with more detailed EPR data for A•1 . 6 most recent PSI structure and the comparison to bRC18 shows that the distance between the two Mg2+ ions and between the planes of the two chlorin rings of P700 are essentially the same as in P960. Thus, the respective primary donors have been superimposed in the two structures. In addition, the membrane normals have been aligned and the helix arrangement has been chosen such that the L side of the bRC structure and the unprimed helices of PSI are superimposed. This places the tentatively assigned quinone position, QK, in PSI on the same side of the RC as QA in bRC. Figure 1 (top) presents a side view (perpendicular to the membrane normal) and (bottom) a view down along the membrane normal. With the chosen alignment it can be seen that the helices dL and dM in bRC and the helices m and m′ in PSI fall nearly on top of each other. Note that the latter helices do not extend as far toward the stromal side of the membrane. This is also consistent with the respective alignment of the histidine ligands to P700 and P865 which requires an appreciable shift of the m helix toward the lumenal side compared to the position of the d helix in bRC. The positions of the quinones differ by 5 Å with A1 lying deeper in the membrane than QA. However, the current resolution of the X-ray PSI structure does not yield positions of the amino acid side groups. Thus we have modeled the m and n helices using the constraints given above. 2.5. Three-Dimensional Modeling Procedures. Helices were modeled using the backbone angles (φ, ψ, ω) ) (-64°, -47°, -177°). These perfect helices were centered on the

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Figure 1. Comparison of the cofactor and helix arrangement of bRC (Rhodopseudomonas Viridis) and PSI (Synechococcus elongatus) in the respective regions of the primary donor, P960 and P700, and the quinone acceptor, QA and QK. For bRC, the helices dL and dM, the cofactors P960, QA, and QB as well as the non-heme iron, Fe2+, are shown along with the histidine residues M-H200 and L-H173, and M-H217 and L-H190 which bind P960 and the non-heme iron, respectively (Brookhaven PDB entry 1prc1). For PSI the helices m, m′, and n′, the quinone position QK and the iron-sulfur cluster FX are shown as given by the 4 Å resolution PSI electron density map.6,18 The view directions are (top) perpendicular to the respective membrane normal n (equivalent to the crystalline c-axis in case of PSI) and parallel to the planes of the Chl constituents of the respective primary donor P; (bottom) along the membrane normal n (the c-axis) from the stroma side. The three thin lines (bottom) represent the line connecting the centers of the Chl constituents of the primary donor P, the line connecting the centers QA and QB for bRC, and a corresponding line through the center of QK (PSI) and the local pseudo-C2 axis (this C2 axis approximately runs through FX and the midpoint of the two Mg atoms of P700), SETOR.49

helical axes that were determined from the intermediate resolution electron density map.6 As outlined above the orientation of the m helix was determined from the experimentally supported His680 ligation to the magnesium ion. A histidine within a helix has two allowed rotamers, which can be called “up” and “down” respectively. In the up rotamer, the side chain points along the helix toward the C-terminal end, and in the down rotamer, the side chain points more toward the N-terminal end. Since it is not known which of the rotamers is present in PSI both configurations were considered (see Figure 3 below). This histidine ligation to the magnesium does not leave much further conformational freedom of helix m. Helix n was modeled similarly. This helix has a hydrophobic and a hydrophilic side. In the model we assumed that the hydrophobic

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Figure 2. Modeling of the binding region of A1 (WHAT IF29). The location of A1 has been adapted from the quinone position referred to as QK •in the 4 Å X-ray structure of PSI.6 The orientation of the A1 headgroup has been obtained from transient EPR studies on P•+ 700A1 in PSI single crystals.9 MOLSCRIPT.50 View directions: (top) Perpendicular to the membrane normal and the crystallographic c-axis, parallel to the axis connecting the two Mg atoms of P700; (bottom) viewed from the stroma along the c-axis. The view directions are the same as for Figure 8 of ref 9 (see text). Selected amino acids with side chain are denoted with their one-letter code.

side faces into the membrane (down in the figures) and the hydrophilic side faces the stroma. Helix o can presently not be modeled reliably if the only information available is its axis and no specific interactions are known that can provide further constraints. However, the axis coordinates6 indicate that the helices m and o come too close to each other in the middle. The sequence motif GlyxxAlaxxxAlaxxxAla is found in helix o (o′). In this case one side of helix o consists of only very small residues, and this side could face helix m for a closer approach.

A 3D database search using the SCAN3D options28 in the WHAT IF program29 revealed only one suitable fragment to use as a template for the m-n loop. Lacking more solid information, this loop was modeled in several ways, and it became clear that the translation along the axis of helix n probably has an error of about 2 Å. Selected models were energy minimized in a vacuum with GROMOS,30 restraining the R-carbons to their initial positions. The models as shown in Figures 2, 3, and 4 have acceptable geometry and energetics and agree with the experimental facts. It should be stressed

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that interactions between amino acid side chains and the naphthoquinone, modeling the quinone acceptor A1, have not been included explicitly. 3. Modeling Results and Consistency with Experimental Data 3.1. Model Structures of the A1 Binding Site. Figure 2 represents one of the possible models viewed from the same directions used for Figure 8 of the preceding paper.9 The top view is perpendicular to the crystalline c-axis (or membrane normal n) and parallel to a line through the magnesium atoms of the two chlorophylls of P700. The bottom view is down the c-axis from the stromal side. The helices m and n are shown, as well as the two P700 magnesium ions, the quinone group, the FX cluster, and the side chains of His680, Tyr696, and Trp697. The precise length of the m-n loop is a matter of discussion. Depending on the model built, Ser692 is still part of helix m, or is just too irregular to be called helical by the DSSP program.13 The same ambiguity is observed for Tyr696 at the beginning of helix n. However, in all models, the sequence GlyArgGly (TrpArgGly in PsaB) is part of the m-n loop. The presence of two glycines precludes any definite statements about the role or location of the central, charged arginine in this loop. With the assumption that the hydrophobic side of helix n is directed toward the interior of the reaction center, Trp697 always faces in this direction. Alternative rotamers for Trp697 will clash with other parts of the model. To illustrate the kind of variability of the possible models, the two which result from using the two rotamers for the His ligand to P700 are shown overlayed in Figure 3. Although helix m is shifted by about half a turn, only minor changes occur in the loop to helix n. More importantly, both models suggest that the plane of the Trp697 side chain is parallel to the quinone group. Although many models are possible, and the margin of error is undoubtedly substantial in the two models shown, it is hard to arrive at a model in which the proximity between this tryptophan side chain and the quinone is absent regardless of which of the His680 rotamers is used. In some of our models Tyr696 also faces the quinone, leading to a particularly close interaction if the quinone would be relocated between the Tyr and Trp residues. However, the models are insufficiently precise to make definite statements about this additional interaction. One of the major differences between PsaA and PsaB is found in the m-n loop. In PsaA the GlyArgGly motif is equally well conserved between different species as the TrpArgGly motif in the corresponding position of PsaB. The close proximity of this loop to the quinone headgroup suggests that this difference (Gly T Trp) may play a role in the distinction of the active and inactive branch of the electron-transfer pathway. 3.2. Comparison to QA Binding Region in bRC. Having obtained a model for the A1 binding site in PSI, it is informative to compare it with the QA site in bRC. This comparison is shown in Figure 4 as a close-up of the respective quinone binding regions. One of the models of the A1 binding site (same as in Figure 2) is shown in the top part of Figure 4 from a slightly different perspective than in Figure 2 to allow a better comparison with the analogous section of the bRC structure1 in the bottom part of Figure 4. A striking feature of the two binding sites is that the near coplanarity of A1 and Trp697 predicted in the PSI model (Figure 4, top) is similar to the arrangement of QA and M-Trp250 in bRC (Figure 4, bottom). This arrangement clearly suggests that Trp-quinone π-π interactions play a role in binding the quinone in both systems.

Figure 3. Comparison of two different models of the A1 binding site. The two models result from the two different rotamers of the His680 side chains which bind P700. MOLSCRIPT.50 The view direction is perpendicular to the membrane normal and rotated around this axis by an angle of -50° with respect to the connecting line between the two Mg centers of the Chl a constituents of P700. MOLSCRIPT.50

However, the dominant interaction between QA and the protein in bRC is known to be the strong H bond between the carbonyl oxygen at ring position 4 in QA and M-His217, which is also a ligand to the nonheme iron, Fe2+. The binding of M-His217 to QA and the nonheme iron is indicated by broken lines in Figure 4 (bottom). A second weaker H bond between the other carbonyl oxygen and the backbone amide of M-Ala260 is of lesser importance in the present context. For PSI a strong H bond to a His residue in helix m can be excluded. First, there are no histidines in this region of the PsaA or PsaB amino acid sequences. Second, the m helices are cut short on the stromal side well below the level of M-His217 of helix dM of bRC. Thus, the main result of the comparison is that π-π bonding to a tryptophan residue is a common feature of the two binding sites but that site structure and H bonding to histidine residues are basically different. The alignment of the transmembrane helices and cofactor locations chosen for Figure 1 includes a consequence which deserves closer consideration. For bRC, it is established that light-induced electron transfer proceeds unidirectionally along the L-branch which is also referred to as the A-branch (for a review, see ref 31). It is generally believed but not confirmed

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Figure 4. Comparison of the binding regions in PSI and bRC. In both representations the respective membrane normal lies in the plane of the paper, and runs from bottom to the top. Upper panel: View onto the modeled binding region of A1 with the viewing direction turned by the angle of +57° with respect to the line connecting the two Mg centers of P700. Lower panel: Binding region of QA in bRC from Rps. Viridis1 with the view direction along the normals of the planes of the BChl constituents of P960 MOLSCRIPT.50

yet experimentally that there is also only one active branch in PSI. Another well-established fact, in this case for both types of reaction centers, is an asymmetric spin density distribution over the primary donor cation. ENDOR experiments on bRC have shown that the spin density on P•+ 865 is located predominantly on the L-half, typically by a ratio of 2:1 in Rb. sphaeroides with similar values in Rps. Viridis. In PSI the asymmetry is even more pronounced and the spin density is 27 It had been located to more than 85% on one-half of P•+ 700. suggested early on that the unidirectionality of the electron

transfer and the asymmetry in the spin density distribution may be related phenomena. However, recent results with bRC modified by extensive site directed mutagenis (for review see ref 32) indicate that large variations in the spin density distribution are possible without appreciable effect on the electron-transfer unidirectionality. Independent of such a correlation, it is clear that for native bRC the active electron transfer path as well as the more spin density carrying BChl half of the primary donor cation are associated with the L side. As a consequence, for the charge-

Quinone Acceptor A1 in Photosystem I •separated state P•+ 865QA , the dipolar vector, zd, which connects the spin density centers of the radical ions, is directed more toward the BChl dimer half ligated to the L-protein (i.e., L-His173 in Figure 1). For PSI, it is not known which protein, PsaA or PsaB, binds the chlorophyll carrying the spin density and whether it is associated with the primed or unprimed helices in the structure (see section 2). It also not clear whether the QK site in the electron density map6 is in the active branch or not. As discussed above, the choices needed to produce Figure 1 have been made such that there is complete analogy between the two reaction centers which places QA (bRC) and A1/QK (PSI) on the same half of the RC. For this choice the quinones are bound to helix dM in bRC and helices m′ and n′ in PSI, whereas the spin density carrying half of the primary donor cations are ligated to helix dL in bRC and by analogy to helix m in PSI, respectively ( i.e., the spin density in P•+ 700 is placed on Chl eC1 in ref 6). The main purpose of this consideration is to note that in this case, the projection of zd into the a,b-plane does not align anymore with the a-axis as concluded from transient EPR and spin-echo data.9,11 However, the orientation of zd relative to the crystalline axes depends on the calibration of the crystal orientation. For this procedure the spectra of FA in a large crystal were used and it was assumed that the c-axis and the a (or b)-axis lie in the same macroscopic plane. Thus, the following alternatives remain: either the assumption used in the calibration procedure is not valid or the asymmetric spin density distribution in P•+ 700 is different than in bRC. Clearly, an independent check of the calibration is required before a definite conclusion about the relative positions on the spin densities in the two systems can be drawn. Although we have chosen the analogy to bRC for the figures in this paper, the decisions as to which is the active branch and the associated protein subunit remain open questions. The A1 binding site model has been constructed using structural data and constraints. However, the magnetic resonance data which are available in comparable quality for both QA in bRC and A1 in PSI also contain information about their electronic structure and binding properties. Again, the comparison of the two types of reaction centers can be used advantageously and provides a further test. 3.3. Influence of the Environment As Reflected in the Magnetic Interactions of A•1 . Reliable correlations between the magnetic resonance data and the characteristics of the quinone environment can be evaluated only if an atomic structure of the cofactor and its binding site is available as is the case for the RC’s of some purple bacteria. In addition, specific modifications of the cofactor (e.g. by substitution) and of the environment (e.g. by site-directed mutagenesis) can be used to augment the information from the native system. Here, we will (i) test our modeling results for consistency with conclusions derived from a wide range of magnetic resonance data and (ii) attempt to draw further conclusions about the A1 binding site by exploiting some established correlations between magnetic interaction parameters and structural properties, available for bRC. 3.3.1. Hyperfine Interactions. In principle, hyperfine couplings (hfc) provide information about the electronic structure of the semiquinone radical at an atomic level because they are associated with the individual nuclear spins. A feature of particular interest which is reflected in the hfc is the molecular asymmetry introduced through the interaction of the quinone with the protein environment, in particular in the form of asymmetric hydrogen bonds to the carbonyl groups. The

J. Phys. Chem. B, Vol. 102, No. 42, 1998 8285 TABLE 1: Comparison of the 1H Hyperfine Coupling (hfc) •Values (MHz) for Q•A and A1 within the Respective Binding Site (bRC and PSI) and in Isotropic Solution (Ip) ubiquinone10 coupling

QAa

phylloquinone Ipb,c

A1d,e

Ipb,d,e

CH3k A| 6.6 8.5 12.7 12.8 9.5 10.0 A⊥ 3.4 4.8 9.3 9.0 6.3 6.8 Aiso 4.5 6.0 10.4 10.3 7.4 7.9 β-CH2l A| 8.7 5.0f g h i A⊥ 3.4 gyy > gzz. Thus, the shift of the xx component of the tensor from the free electron value, gxx - ge, provides a measure of the interaction of the quinone with its environment. In particular, the change in this shift ∆(gxx - ge) between isotropic frozen polar solution and bRC or PSI binding site can be used to compare the different environments. A selection of high-accuracy experimental g anisotropies is shown in Table 2. In view of the difficulties in interpreting such data, we will restrict ourselves to a qualitative discussion of the tendencies in the g anisotropy. Based on the perturbation treatment by Stone,35,36 gxx - ge is expected to decrease with increasing H-bond strength and to increase with stronger π-π bonding.37 Thus, it is likely that these two effects will compensate one another. This appears to be the case when the values for ubiquinone in the QA and QB binding sites and in 2-propanol are compared. Very little difference in all g-tensor elements is observed, although it is known that the H bonding in the three environments is significantly different and is particularly strong for Q•A . On the other hand, the ∆(gxx - ge) value shown in Table 2 for A1 is considerably larger than the value for QA. In terms of the expected tendencies, this can be taken as evidence that the quinone acceptor in PSI experiences weaker H bonding and/or stronger π-π bonding to the protein environment in comparison to bRC. This effect is even more pronounced when the native quinone (ubiquinone or phylloquinone) is substituted with naphthoquinone or duroquinone.37 Moreover, the orientation of the substituted quinones in the PSI reaction center is changed drastically,37,38 although substitution has been confirmed to occur into the same structural site.39 Neither the g anisotropy nor the orientation of the quinones changes significantly when the corresponding substitutions are carried out in bRC.37 The latter result is highly plausible if strong H bonds are responsible for keeping the quinones bound in the QA site of bRC. In contrast, the easier solvent extractability and exchangeability of A1, the different orientations of the substituted quinones, and the larger g anisotropies in PSI all suggest that strong H bonds are not likely in the A1 site. 4. Discussion Certainly, the facts and modeling results compiled here suggest that the TyrTrp pair, located at the start of the surface helix n of PSI, is an important element for the binding of the A1 acceptor in PSI. Indeed, it is well conserved both between

PsaA and B and in all known sequences of PSI from either plants or cyanobacteria. However, the TyrTrp pair has not been found in the corresponding protein sequences of green sulfur bacteria and heliobacteria,40,41 which also belong to the type I reaction centers. Interestingly, the role of the quinones in these systems is not firmly established and there is considerable amount of data which suggests that they may not be involved in forward electron transfer; see e.g. ref 42. Although alignment of the quinone plane of QA with that of a nearby tryptophan residue is a clear feature in the bRC structure (see Figure 4), the significance of this π-π interaction for QA binding is not so obvious, because hydrogen bonding plays the predominant role. However, as discussed above, the g-tensor values suggest π-π bonding is stronger for A1 in PSI, perhaps even partly compensated by some weak H bonding as indicated in the ENDOR experiments.43-45 The postulated interaction between A1 and a tryptophan residue is experimentally confirmed in ESEEM experiments.46 In addition, weak evidence has been suggested for another coupling to a His residue.46 However, the proposed residue (PsaA-His708, Scheme 2) is located toward the end of helix n and is therefore too remote for significant interaction with A1. However, an interesting alternative His candidate is available in helix o (PsaA-His734, Scheme 2). Note that the distance of this residue from the stromal end of the helix o corresponds roughly to the distance of the corresponding His residue (M-His264) in helix e of bRC. Its function in bRC is ligation of the nonheme iron (see Figure 4, bottom). PSI lacks the nonheme iron, and hence this His residue would be available for other purposes. Its location would permit some interaction with A1. If correct, it would represent an interesting case where a proteinwise similarly positioned His residue would serve with a completely different function in the two types of RC’s. 5. Conclusions Although, the models of the A1 binding site shown in Figure 2, 3, and 4 have some degree of error and conclusions based on them should be treated with appropriate caution, they show that there are no internal inconsistencies in the experimental data because all data can be reconciled in the proposed models with acceptable stereochemistry and energetics. Given the fact that (i) it is almost impossible to build a model in which Trp697 does not interact with the quinone, (ii) a quinone-tryptophan interaction is present in bRC, and (iii) EPR and ENDOR data are consistent with such an interaction, the conclusion that this interaction is important for A1 binding is quite reliable. Obviously, these modeling results and proposed proteinquinone interactions suggest specific mutation experiments. The TyrTrp pair has already been targeted for some time. Other structurally relevant residues near the m,n loop are also of interest. Finally, the His residue in the helix o is also an interesting candidate. Note Added in Proof. For the figures of this paper the angle between the planes (c,zd) and of the A•1 quinone head group

Quinone Acceptor A1 in Photosystem I has been assumed to be zero, although it is only specified within an upper limit of 60° according to the structural model presented in the preceding paper.9 This uncertainty (with respect to the rotation of A•1 around the gxx/zd axis) could be removed by recent EPR/ENDOR data. Preliminary evaluation determines the angle between the two planes as close to 60°. The conclusions from our modeling study, in particular concerning the Trp-quinone interaction, are not affected because the rotational freedom of the Trp ring still permits coplanarity with the adjusted A•1 quinone plane. Acknowledgment. We are grateful to W.-D. Schubert, O. Klukas, and N. Krauss (FU Berlin, Germany) for providing the coordinates of the central axes of helices m, n, and o, of the Mg atoms of P700 and of the center of FX, for stimulating discussions, and, in particular, for preparation of Figure 1. We especially want to thank Gert Vriend (EMBL Heidelberg) for his invaluable support with the computer modeling and many helpful suggestions. This work was supported by the Deutsche Forschungsgemeinschaft (SfB 312; Teilprojekte A1, A3 and A4), NaFo¨G (graduate fellowship to S.G.Z.), and Fonds der Chemischen Industrie (to W.L.). References and Notes (1) (a) Abbreviations: bRC, purple bacteria reaction center (RC); PSI and PSII, Photosystem I and II; Chla, chlorophyll-a; VK1, vitamin K1 or phylloquinone; A1, quinone electron acceptor in PSI, a VK1 molecule, also termed QK; QA and QB, primary and secondary electron acceptors in bRC, respectively; P700 and P865, primary donor (B) CHl a dimer in PSI and bRC, respectively; FX, FeS in PSI; zd, dipolar coupling vector between P•+ 700 and A•1 ; PsaA and PsaB, RC proteins of PSI; L and M, RC proteins of bRC; EPR, electron paramagnetic resonance; ENDOR, electron nuclear double resonance; ESEEM, electron spin-echo envelope modulation; PHD, profilebased neural network prediction of secondary structure. (b) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. Nature 1985, 318, 618. Brookhaven Protein Data Bank, entry 1prc. (2) Ermler, U.; Fritzsch, G.; Buchanan, S. K.; Michel, H. Structure 1994, 2, 925. Brookhaven Protein Data Bank, entry 1pcr. (3) Stowell, M. H.; McPhillips, T. M.; Rees, D. C.; Soltis, S. M.; Abresch, E.; Feher, G. Science 1997, 276, 812. Brookhaven Protein Data Bank, entries 1aij and 1aig. (4) Krauss, N.; Hinrichs, W.; Witt, I.; Fromme, P.; Pritzkow, W.; Dauter, Z.; Betzel, C.; Wilson, K.; Witt, H. T.; Saenger, W. Nature 1993, 361, 326. (5) Krauss, N.; Schubert, W.-D.; Klukas, O.; Fromme, P.; Witt, H. T.; Saenger, W. Nature, Struct. Biol. 1996, 3, 965. (6) Schubert, W.-D.; Klukas, O.; Krauss, N.; Saenger, W.; Fromme, P.; Witt, H. T. J. Mol. Biol. 1997, 272, 741. (7) Pierson, B.; Olson, J. In Microbiological Materials; Cohen, Y., Rosenberg, E., Eds.; American Society for Microbiology; Washington, DC; 1989; pp 402-427. (8) Blankenship, R. E. Photosynth. Res. 1992, 33, 91. (9) Kamlowski, A.; Zech, S. G.; Fromme, P.; Bittl, R.; Lubitz, W.; Stehlik, D. J. Phys. Chem. B 1998, 102, 8266. (10) MacMillan, F.; Hanley, J.; van der Weerd, L.; Knu¨pling, M.; Un, S.; Rutherford, A. W. Biochemistry 1997, 36, 9297. (11) Bittl, R.; Zech, S. G.; Fromme, P.; Witt, H. T.; Lubitz, W. Biochemistry 1997, 36, 12001. (12) Yang, F.; Shen, G.; Zybailov, W. S. B.; Ganago, A.; Bryant, D.; Golbeck, J. J. Phys. Chem. B 1998, 102, 8288. (13) Kabsch, W.; Sander, C. FEBS Lett. 1983, 155, 179. (14) Rost, B.; Sander, C. J. Mol. Biol. 1993, 232, 584. (15) Rost, B.; Casadio, R.; Fariselli, P.; Sander, C. Protein Sci. 1995, 4, 521. (16) Mu¨hlenhoff, U.; Haehnel, W.; Witt, H. T.; Herrmann, R. G. Gene 1993, 127, 71. SWISS-PROT P25896 (PsaA) P25897 (PsaB).

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