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Photosystem II (PSII) is that part of thephotosynthetic apparatus that uses lightenergy to split water and evolve oxy-gen1. Understanding the molecular detailsof this reaction is one of the great chal-lenges of molecular cell biology. In a recent article in TiBS, Rgner et al.2 pro-posed a model for the positioning of various subunits of the PSII complex.Their model was, in part, based on elec-tron microscopy and difference map-ping of isolated PSII complexes that hadvarious subunit compositions3. Sincethen, we and colleagues46 have gener-ated new structural data and projectionmaps of substantially higher resolutionby electron crystallography. These stud-ies have revealed the location of themajor subunits of the PSII core and, inmany cases, the organization of theirtransmembrane helices. Here, we sum-marize these newly available structuraldetails to produce a greatly improvedmodel of PSII. The new structural modelhighlights the evolutionary links betweenthe reaction centres of PSII, photosys-tem I (PSI) and purple bacteria, whichother studies713 have suggested, andhas important implications with respectto the mechanism of D1 turnover and excitation-energy transfer.

PSII is a multisubunit protein complexthat is located in the photosyntheticmembranes of plants, algae and cyano-bacteria14,15. We isolated and character-ized (structurally and biochemically) a large dimeric complex that containsthe majority of the spinach PSII sub-units3,16,17. Because the complex containsthe light-harvesting chlorophyll abcomplex (LHCII) as well as the PSII core-reaction-centre proteins, it has beentermed the LHCIIPSII supercomplex3,10.The structure of this isolated complexserved as the framework for positioningof various protein subunits in the previ-ous model of PSII (Ref. 2). In particular,the model suggested the possible lo-cations of two inner-antennae subunits,CP47 and CP43, relative to those of the re-action-centre proteins, D1 and D2. In thelight of new results46, we must reassessthe relative positions of these four sub-units and improve the overall resolutionof the model.

Positioning of the major intrinsic subunitswithin the photosystem II core

Single-particle analysis3 has shownthat the LHCIIPSII supercomplex con-tains a centrally located dimeric PSII core,which is flanked by two sets of chloro-phyll a- and chlorophyll b-binding pro-teins. The major intrinsic subunits of thePSII core can be divided into two func-tional groups. The first group consists ofthe reaction-centre proteins, D1 and D2,which bind the cofactors needed for pri-mary and secondary charge separation1.D1 and D2 are encoded by the PsbA andPsbD genes, respectively, have similarmolecular weights (38.0 kDa and 39.4kDa, respectively) and each is predictedto contain five transmembrane helices(Fig. 1b). The D1 and D2 subunits inter-act intimately with each other to form aheterodimer. The second functional groupof intrinsic proteins is the inner-antennachlorophyll-a-binding proteins, CP47 andCP43, which are encoded by the PsbBand PsbC genes, respectively. These alsohave similar molecular weights (56.3 kDaand 50.1 kDa, respectively) and sharesignificant sequence homology; structurepredictions suggest that each containssix transmembrane helices and that their

N- and C-termini are exposed at the stro-mal surface (Fig. 1b)18. Both proteins arecharacterized by the presence of largehydrophilic loops that join transmem-brane helices five and six (Fig. 1b).

The model presented previously2 sug-gested that CP47 is located adjacent tothe D1D2 heterodimer and that CP43 issandwiched between LHCII and CP47.This would mean that CP43 serves as aconduit for the transfer of excitation en-ergy from LHCII to CP47 and, ultimately,to the D1D2 heterodimer. On the basisof new structural data, the assignmentof CP43 and CP47 to the same side of theheterodimer has to be revised. It is nowapparent that CP43 and CP47 must be lo-cated on either side of the D1D2 reac-tion centre and are likely to be related toeach other by the same pseudo-twofoldsymmetry axis around which the D1 andD2 proteins are organized.

The revised arrangement of the CP47,D2, D1 and CP43 subunits arises fromseveral lines of evidence1012 and par-ticularly from the most-recent structuraldata for PSII and PSI. X-ray crystallo-graphic analysis of PSI has reached thestage at which we can identify a-helicalregions as well as chromophores7,8. PSIis composed of two homologous reac-tion-centre proteins (encoded by thePsaA and PsaB genes), each predicted tocontain 11 transmembrane helices (seeFig. 1a). The assignment of these heliceswithin the PSI map led to the surprisingobservation that the five transmem-brane helices at the C-termini of thePsaA and PsaB proteins together form a

FRONTLINES

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Figure 1The transmembrane helices of the major intrinsic proteins of the two photosystems. (a) Photosystem I (PSI). (b) Photosystem II (PSII). Helices that share structural similarityare indicated. Reaction-centre helices are shown in red; helices of the inner antenna areshown in green.

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44

ten-helix cluster, whose organization isstructurally similar to the L and M sub-units in the reaction centre of purplephotosynthetic bacteria. This unexpectedanalogy becomes even more interestingif we consider the similarity betweenthe D1 and D2 proteins in PSII and thebacterial L and M subunits. It was notedmany years ago that the plant and bac-terial proteins share sequence similarity9;more recently, Rhee et al.5 confirmed thatthe proteins have similar structures by electron crystallography. These ob-servations establish a very importantevolutionary link between all types ofphotosynthetic reaction centre5,13.

The similarity between PSI and PSIIhas also turned attention to the possibilitythat the six remaining N-terminal helicesof the PSI reaction-centre proteins mightbe analogous to the six putative trans-membrane segments of CP43 and CP47.Vermaas10, and Rutherford and Nitschke11,had already raised this possibility, but it

was Krauss and co-workers12,13 who em-phasized the fact that the various sharedweak sequence homologies could meanthat these PSII and PSI proteins are simi-lar structurally. Given that the PsaA andPsaB reaction-centre proteins of PSI arerelated to each other by a pseudo-twofold symmetry axis, CP43 and CP47might also be related by the pseudo-twofold symmetry axis around whichthe D1 and D2 proteins are arranged(see Fig. 1).

We and co-workers5 have obtained an8- resolution, three-dimensional mapof the isolated CP47D1D2 subcorecomplex by electron crystallography.This map, coupled with other recentcryoelectron-microscopy data4,6, hasprovided definitive experimental sup-port for the proposal that PSII and PSIshare structural similarity and shownthat the helices of CP43 and CP47 arestructurally analogous to the six trans-membrane helices of the PsaA and PsaB

reaction-centre proteins of PSI. Usingthis information, we can construct anew model for the positioning of the D1,D2, CP47 and CP43 proteins within thePSII core (see Fig. 2a). The positioning ofthese proteins is, in part, based on a 16-projection map of a dimeric PSII-corecomplex derived from cryoelectron mi-croscopy6. The ten transmembrane he-lices in the D1 and D2 heterodimer areflanked by the helices of CP47 and CP43.We calculated the positions of the D1,D2 and CP47 transmembrane helicesfrom results given in Ref. 5; we inferredthe positions of the CP43 transmem-brane helices by drawing analogies withthe PSI structure13 and from the datapresented in Refs 5 and 6. In our model,the D1 and D2 proteins are adjacent toCP43 and CP47, respectively. The rea-soning for this is based on evidence thatCP43 becomes dislodged from the PSII-core complex during turnover of the D1protein19. The seven helices shown in

Figure 2(a) Helix organization of major intrinsic proteins of the photosystem II (PSII) core. The helix organization of the D1 and D2 proteins (shown in red)and of CP47 (shown in green) derives from an 8- three-dimensional structure of the CP47D1D2 subcore complex5. The six helices of CP43(shown in green) have been superimposed on the assumption that they are identical to those of CP47 and are related to them by a pseudo-twofold-symmetry axis that is shared with the D1 and D2 proteins. The seven additional helices (shown in grey) were identified in theCP47D1D2 subcomplex5. The framework for the model is a 16- map of the dimeric PSII-core complex that was obtained by cryoelectron mi-croscopy6. (b) Subunit positioning of major proteins of the light-harvesting complex II (LHCII)PSII supercomplex. The helices of CP47, CP43 andthe D1D2 heterodimer are positioned as in (a). The positions of the transmembrane and surface helices of the LHCII trimer, CP29 and CP26 arebased on structural data21 and sequence homologies23. Their positions are in part based on the previous model2 and on recent crosslinkingdata22. Difference mapping by single-particle analysis of negatively stained preparations3,16 has revealed the positions of the 33-kDa and 23-kDaextrinsic proteins (shown in orange and yellow, respectively) of the oxygen-evolving complex. Both (a) and (b) are viewed from the luminal side. (c) Side view of the negatively stained LHCIIPSII supercomplex that identifies protrusions due to the 33-kDa and 23-kDa extrinsic proteins.

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grey in Fig. 2a were also identified in the8- map of the CP47D1D2 subcorecomplex5 and probably belong to thePsbE, PsbF, PsbI, PsbK, PsbL, PsbT andPsbW proteins20, but have not yet beenindividually assigned. The core is likelyto contain additional low-molecular-weight intrinsic proteins (e.g PsbH,PsbJ, PsbM and PsbN), but visualizationof their helices has yet to be achieved.

Positioning of the oxygen-evolving complexand outer-antenna proteins within theLHCIIPSII supercomplex

Using the model presented in Fig. 2a,we can position the D1, D2, CP47 andCP43 helices on the top-view projectionmap of the LHCIIPSII supercomplex de-rived from single-particle analysis ofnegatively stained preparations (Fig.2b). We have used difference mapping ofisolated PSII complexes by the single-particle approach to localize the 33-kDaand 23-kDa extrinsic proteins of the oxy-gen-evolving complex3,16. The arrange-ment of the three transmembrane he-lices and one luminal surface helix ofthe LHCII trimer is based on the pub-lished 3.4- structure21; the helices arelocated as previously suggested2. Thepositioning of the minor LHCII-like pro-teins, CP29 and CP26, relative to CP47and CP43 is based on recent findings22;these proteins are depicted as having

three transmembrane-spanning regionsthat are similar to those of LHCII (Ref.23). This model is consistent with a con-siderable amount of crosslinking data15

and should serve as a basis for elucidat-ing further the molecular processes thatunderlie photosynthetic water splittingand oxygen evolution.

AcknowledgementsWe thank colleagues with whom we

have collaborated (Egbert Boekema,Matthias Rgner, Werner Khlbrandtand Kyong-Hi Rhee) or had discussions(Jan Dekker, Petra Fromme, RobertoBassi, Bill Rutherford, Wim Vermaas andBertil Andersson). J. B. thanks theBBSRC for financial support.

References1 Diner, B. and Babcock, J. (1996) in Oxygenic

Photosynthesis: The Light Reactions (Ort, D. R.and Yocum, C. F., eds), pp. 213247, KluwerAcademic Publishers

2 Rgner, M., Boekema, E. J. and Barber, J.(1996) Trends Biochem. Sci. 21, 4449

3 Boekema, E. J. et al. (1995) Proc. Natl. Acad.Sci. U. S. A. 92, 175179

4 Rhee, K-H. et al. (1997) Nature 389, 5225265 Rhee, K-H., Morris, E. P., Barber, J. and

Khlbrandt, W. (1998) Nature 396, 2832866 Hankamer, B., Morris, E. P. and Barber, J. in

Proceedings of the XIth International Congresson Photosynthesis, Kluwer Academic Publishers(in press)

7 Krauss, N. et al. (1996) Nat. Struct. Biol. 3,965973

8 Schubert, W-D. et al. (1977) J. Mol. Biol. 272,

7417699 Michel, H. and Deisenhofer, J. (1988)

Biochemistry 27, 1710 Vermaas, W. F. J. (1994) Photosynth. Res. 41,

28529411 Rutherford, A. W. and Nitschke, W. (1996) in

Origin and Evolution of Biological EnergyConversion (Baltscheffsky, H., ed.), pp. 143174, VCH, New York

12 Fromme, P. et al. (1996) Biochim. Biophys. Acta1275, 7683

13 Schubert, W-D. et al. (1998) J. Mol. Biol. 280,297314

14 Barber, J. et al. (1997) Physiol. Plant. 100,817827

15 Hankamer, B., Barber, J. and Boekema, E. J.(1997) Annu. Rev. Plant Physiol. Mol. Biol. 48,641671

16 Boekema, E. J., Nield, J., Hankamer, B. andBarber, J. (1998) Eur. J. Biochem. 252,268276

17 Hankamer, B. et al. (1997) Eur. J. Biochem.243, 422429

18 Bricker, T. M. (1990) Photosyn. Res. 24, 11319 Barbato, R. et al. (1992) J. Cell Biol. 119,

32533520 Zheleva, D. et al. (1998) J. Biol. Chem. 273,

161221612721 Khlbrandt, W., Wang, D. N. and Fujiyoshi, Y.

(1994) Nature 367, 61462122 Harrer, R., Bassi, R., Testi, M. G. and Shafer, C.

(1998) Eur. J. Biochem. 255, 19620523 Green, B. R., Pichersky, E. and Kloppstech, K.

(1991) Trends Biochem. Sci. 16, 181186

JAMES BARBER, JON NIELD, EDWARD P. MORRIS AND BEN HANKAMER

The Wolfson Laboratories, Biochemistry Dept,Imperial College of Science, Technology andMedicine, London, UK SW7 2AY.

OBITUARY

Samuel D. Weiss (Fig. 1), the discovererof RNA polymerase, died in Ancona,Italy on 1 December 1997. He had re-tired from the Dept of Biochemistry atthe University of Chicago in 1992, hav-ing been on the faculty since 1958.Although he did all of his work on RNApolymerase in Chicago, Sam was a NewYorker by birth. He graduated from CityCollege in 1948 (after serving in the USArmy from 1943 to 1946) and then wentwest to the University of SouthernCalifornia for his graduate work oncholesterol synthesis and metabolism.Following the completion of his PhDthesis in 1954, he moved to Chicago forthe first time, as a postdoctoral fellowwith Eugene Kennedy, to study thebiosynthesis of lecithin. A story from

this early phase of his career is typicalof Sams approach to science. He sus-pected that there was a critical but un-recognized factor in the ATP used in thein vitro system for lecithin biosynthesis.His careful analytical work revealed thefactor to be CTP, which often contami-nated commercial ATP in those days.This discovery led immediately to theidentification of CDP-choline, whichEugene Kennedy synthesized andshowed to be a new intermediate inlecithin biosynthesis1. This was the firstdemonstration of the role of cytidine-containing intermediates in the synthe-sis of phospholipids and triglycerides.

Having decided to continue with fur-ther postdoctoral training, Sam joinedFritz Lipmanns group, which was, at

that time, struggling to understand cer-tain aspects of the chemistry of proteinsynthesis. When the Lipmann groupmoved from the Massachusetts GeneralHospital to Rockefeller University in1957, Sam returned to New York, con-tinuing his studies of the activation ofamino acids and the role of transferRNA in protein synthesis2. This work,and his earlier studies, convinced himthat ribonucleoside triphosphates hadto be the true precursors of RNA and thus parallel deoxyribonucleosidetriphosphates, which Arthur Kornbergsgroup had shown to be the true precur-sors of DNA. In 1958, Sam returned tothe University of Chicago to take up aposition as an Assistant Professor ofBiochemistry. His laboratory was in theArgonne Cancer Research Hospital,which for many years was run by theUniversity of Chicago for the Dept ofEnergy; there Sam had the freedom tofollow his imaginative research programwithout the constraint of having to per-form routine experiments to justify anannual research budget.

Professor Samuel D. Weiss,19261997