Three-dimensional Structure of Chlamydomonas reinhardtii and Synechococcus elongatus Photosystem II Complexes Allows for Comparison of Their Oxygen-evolving Complex Organization

Abstract

Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green algaChlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus. The analyses have yielded three-dimensional structures at 30-Å resolution. Biochemical analysis of the C. reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII)·PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure. Not only was the C. reinhardtii LHCII·PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins. The particle isolated from S. elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochromec 550), which were again visualized as protrusions on the lumenal surface of the complex. The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography. By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C. reinhardtii and S. elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins. It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein. However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface. Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins ofC. reinhardtii and S. elongatus are similar and consistent with those determined previously for the OEC proteins of spinach. Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green algaChlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus. The analyses have yielded three-dimensional structures at 30-Å resolution. Biochemical analysis of the C. reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII)·PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure. Not only was the C. reinhardtii LHCII·PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins. The particle isolated from S. elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochromec 550), which were again visualized as protrusions on the lumenal surface of the complex. The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography. By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C. reinhardtii and S. elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins. It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein. However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface. Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins ofC. reinhardtii and S. elongatus are similar and consistent with those determined previously for the OEC proteins of spinach. photosystem II three-dimensional light-harvesting complex oxygen-evolving complex cytochrome polyacrylamide gel electrophoresis Photosystem II (PSII)1is a large multisubunit protein complex that utilizes light energy to split water into molecular oxygen and reducing equivalents. It is located in thylakoid membranes of plants, algae, and cyanobacteria. To understand the molecular mechanisms of PSII requires knowledge of its 3D structure and particularly of the catalytic center, which is responsible for bringing about the thermodynamically difficult reactions of water oxidation and oxygen evolution. In its complete form the PSII complex is composed of over 25 different proteins (1Barber J. Nield J. Morris E.P. Zheleva D. Hankamer B. Physiol. Plant. 1997; 100: 817-827Crossref Google Scholar). At the heart of this multisubunit complex is the reaction center composed of the D1 and D2 proteins, which are encoded by thepsbA and psbD genes. These proteins each consist of five transmembrane helices and bind the redox-active cofactors responsible for the primary and secondary electron transfer processes that occur following the absorption of a photon (1Barber J. Nield J. Morris E.P. Zheleva D. Hankamer B. Physiol. Plant. 1997; 100: 817-827Crossref Google Scholar, 2Diner B.A. Babcock G.T. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: the Light Reactions. Kluwer, Dordrecht, The Netherlands1996: 213-247Google Scholar). It is also likely that the manganese cluster, which catalyzes the oxidation of water, is ligated to the lumenal side of the D1 protein (3Nixon P.J. Diner B.A. Biochem. Soc. Trans. 1994; 22: 338-343Crossref PubMed Scopus (61) Google Scholar). Surrounding the D1 and D2 proteins are the other PSII subunits. These include the chlorophyll a binding proteins CP47 and CP43 that transfer excitation energy to the reaction center (4Bricker T.M. Photosynth. Res. 1990; 24: 1-13Crossref PubMed Scopus (192) Google Scholar). They are encoded by the psbB and psbC genes, respectively, and both proteins have six transmembrane helices characterized by very large loops joining the lumenal ends of helices five and six. A structural model for the organization of the transmembrane helices of these PSII subunits has been deduced from electron crystallography (5Rhee K.-H. Morris E.P. Zheleva D. Hankamer B. Kühlbrandt W. Barber J. Nature. 1997; 389: 522-526Crossref Scopus (127) Google Scholar, 6Rhee K.-H. Morris E.P. Barber J. Kühlbrandt W. Nature. 1998; 396: 283-286Crossref PubMed Scopus (284) Google Scholar, 7Hankamer B. Morris E.P. Barber J. Nat. Struct. Biol. 1999; 6: 560-564Crossref PubMed Scopus (116) Google Scholar), which also revealed the transmembrane helices of other low molecular weight subunits of the PSII core. Higher plants and green algae have an additional outer light-harvesting system composed of proteins that bind both chlorophyll a and b, of which there are six types (Lhcb proteins) (8Jansson S. Biochim. Biophys. Acta. 1994; 1184: 1-19Crossref PubMed Scopus (592) Google Scholar). The majority of the light-harvesting systems are made up of Lhcb1–3 and are known as light-harvesting complex II (LHCII). LHCII is organized as trimers, the structure of which has been determined to 3.4 Å (9Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1541) Google Scholar). The other chlorophyll a/b-binding proteins, Lhcb4, Lhcb5, and Lhcb6, also known as CP29, CP26, and CP24, respectively, bind less chlorophyll b than LHCII and are thought to exist as monomers. In cyanobacteria and red algae the outer light-harvesting system is contained in phycobilisomes attached to the stromal surface of the core complex. They consist of phycobiliproteins, such as allophycocyanin, phycocyanin, and phycoerythrin, which are encoded byapc and cpc genes (10Glazer A.N. J. Appl. Phycol. 1994; 6: 105-112Crossref Scopus (322) Google Scholar). In all types of oxygenic photosynthetic organisms the manganese cluster is stabilized by an extrinsic OEC protein encoded by thepsbO gene, having an apparent molecular mass of 33 kDa. There is no evidence to suggest that the manganese cluster is directly ligated to this protein and, indeed, it can be removed without inhibiting the water oxidation process (11Murata M. Miyao N. FEBS Lett. 1985; 180: 303-308Crossref Scopus (136) Google Scholar, 12Mayes S.R. Cook K.M. Self S.J. Zhang Z. Barber J. Biochim. Biophys. Acta. 1991; 1060: 1-12Crossref Scopus (106) Google Scholar). In plants and algae, two other OEC extrinsic proteins are found, PsbP (23 kDa) and PsbQ (17 kDa). These two proteins play a role in optimizing the levels of Ca2+ and Cl−, which are required for the water-splitting reaction (13Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1085) Google Scholar). In cyanobacteria, however, these proteins are replaced by PsbU (12 kDa) and PsbV (15 kDa), where the latter is cytochrome c 550 (Cytc 550) (14Shen J.R. Ikeuchi M. Inoue Y. FEBS Lett. 1992; 301: 145-149Crossref PubMed Scopus (121) Google Scholar). It seems that these two cyanobacterial proteins also function to create the correct Ca2+ and Cl− environment for the water-oxidation process although the presence of a redox-active cytochrome suggests some other additional function. In this paper we report 3D structural maps of PSII complexes isolated from the green alga Chlamydomonas reinhardtii and the cyanobacterium Synechococcus elongatus. The structures have been obtained by electron microscopy and single-particle analyses of negatively stained preparations and reveal the location of the extrinsic OEC proteins on the lumenal surface of the two types of PSII complex. By incorporating the higher-resolution structure of the underlying intrinsic proteins derived from electron crystallography (5Rhee K.-H. Morris E.P. Zheleva D. Hankamer B. Kühlbrandt W. Barber J. Nature. 1997; 389: 522-526Crossref Scopus (127) Google Scholar, 6Rhee K.-H. Morris E.P. Barber J. Kühlbrandt W. Nature. 1998; 396: 283-286Crossref PubMed Scopus (284) Google Scholar, 7Hankamer B. Morris E.P. Barber J. Nat. Struct. Biol. 1999; 6: 560-564Crossref PubMed Scopus (116) Google Scholar) into the 3D models we have been able to compare the organization of the OEC proteins for algal and cyanobacterial PSII. The C. reinhardtii mutant Xba9, deficient in photosystem I, was grown in Tris acetate phosphate medium (15Harris E.H. The Chlamydomonas Sourcebook. Academic Press, San Deigo, CA1989: 576-577Google Scholar), including 25 μg ml−1 spectinomycin, 100 μg ml−1 ampicillin, and 10 μm3-(3,4-dichlorophenyl)-1,1-dimethyl urea (16Andronis C. Kruse O. Deak Z. Vass I. Diner B.A. Nixon P.J. Plant Physiol. 1998; 117: 515-524Crossref PubMed Scopus (22) Google Scholar). The cells were washed and broken by French press (4,000 p.s.i.), and the thylakoid membranes were collected by sucrose density cushion centrifugation according to the method described by Diner and Wollman (17Diner B.A. Wollman F.-A. Eur. J. Biochem. 1980; 110: 521-526Crossref PubMed Scopus (114) Google Scholar). LHCII·PSII supercomplexes were purified from the thylakoids by continuous sucrose density gradient centrifugation, modified from that previously described (18Hankamer B. Nield J. Zheleva D. Boekema E.J. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (173) Google Scholar) at the final solubilization step, where 50 mm β-d-dodecyl-maltoside was added instead of 20 mm. The LHCII·PSII supercomplexes corresponded to the densest fraction of the gradient. S. elongatus cells were grown as a 30-liter culture in an airlift fermentor using medium D as described by Castenholz (19Castenholz R.W. Bacteriol. Rev. 1969; 33: PubMed Google Scholar), at which in was The culture was a continuous light of PSII were purified from S. a modified from that described previously Hankamer B. C. Barber J. Photosynthesis: and Academic Dordrecht, The Scholar), to The of thylakoids was on the described by and Scholar) and of an in the presence of and of the cells by French press by thylakoids were obtained by centrifugation and were washed to PSII was from the washed thylakoids and purified on a PSII and were by centrifugation on sucrose density The protein of PSII preparations was by The were stained using stained using a method O. Zheleva D. Barber J. FEBS Lett. 1998; Scopus Google Scholar). were using an were negatively stained using the method with A electron was at the at a of and of for the S. elongatus PSII and for the C. reinhardtii were for and The were using a of of at a size of 20 Å on the was within the environment M. M. J. Struct. Biol. PubMed Scopus Google Scholar), the system on a the S. a data of single-particle was obtained by all that were in with other and each was into a A similar data of was from obtained from the C. reinhardtii PSII supercomplex each into a analysis of these two data was made with the M. J. PubMed Scopus Google Scholar). of in size and shape in each The were in as data with the the for M. J. M. J. Struct. Biol. PubMed Scopus Google Scholar), to with an to were determined for the by the M. PubMed Scopus Google Scholar), in 3D from of the projection M. J. M. J. Struct. Biol. PubMed Scopus Google Scholar). were from each 3D model and to additional and to the within each data At each of the were using this the data to the 3D The resolution was determined by two 3D M. J. M. J. Struct. Biol. PubMed Scopus Google Scholar). data derived from the K.-H. Morris E.P. Barber J. Kühlbrandt W. Nature. 1998; 396: 283-286Crossref PubMed Scopus (284) Google Scholar) and PSII core B. Morris E.P. Barber J. Nat. Struct. Biol. 1999; 6: 560-564Crossref PubMed Scopus (116) Google Scholar) maps were into the two using the M. 1991; PubMed Scopus Google Scholar). The B. M. J. Struct. Biol. PubMed Scopus Google Scholar) was to the 3D maps in and is a 3D of the S. core complex at Å from an to the lumenal surface and the extrinsic OEC proteins to the PsbO kDa) and to the PsbU (12 kDa) and PsbV c 550 (15 kDa) The and as described and to PSII preparations from C. reinhardtii and S. retained OEC proteins. The C. reinhardtii complex contained chlorophyll b, as as and its absorption was to that of the LHCII·PSII supercomplex previously isolated from spinach (18Hankamer B. Nield J. Zheleva D. Boekema E.J. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (173) Google Scholar). its protein as determined by is very similar to that of the spinach LHCII·PSII supercomplex (18Hankamer B. Nield J. Zheleva D. Boekema E.J. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (173) Google Scholar). The absorption of the S. in b with that of the isolated C. reinhardtii as it have chlorophyll b absorption The polyacrylamide gel electrophoresis gel of the S. elongatus is also in The that the algal and cyanobacterial PSII complexes OEC proteins and that the PsbP (23 kDa) and PsbQ (17 kDa) of C. reinhardtii are replaced by PsbU (12 kDa) and PsbV c kDa) in S. whereas the PsbO kDa) protein is to The C. were negatively stained and using a electron a data of were by and to to were and relative were determined by the a five algal PSII supercomplex of the in for the final 3D which is in c with its stained S. elongatus PSII dimeric core were also using a electron and an data of about single-particle was with C. the of the S. elongatus were by two described as and side was to be on the electron microscopy were and the data was to to the of the the final 3D were that the of of these are in and the from the final 3D are in b of the for of the 3D structure of the C. reinhardtii supercomplex is in In its it is similar to that for the isolated spinach LHCII·PSII that in the latter the 3D model was obtained by microscopy at a resolution of Å J. Morris E.P. B. M. Barber J. Nat. Struct. Biol. PubMed Scopus Google Scholar). The model for the C. reinhardtii supercomplex is at a resolution to and is attributed to the presence of and its with E.J. Hankamer B. D. J. Nield J. Barber J. M. S. PubMed Scopus Google Scholar). The supercomplex is having been to be thus two of subunits. The protrusions on the lumenal and in are the OEC proteins on with those on the lumenal surface of the spinach PSII supercomplex J. Morris E.P. B. M. Barber J. Nat. Struct. Biol. PubMed Scopus Google Scholar). have been in the spinach complex as the extrinsic PsbO kDa) whereas the protrusions are thought to both the PsbP (23 kDa) and PsbQ (17 kDa) proteins J. Morris E.P. B. M. Barber J. Nat. Struct. Biol. PubMed Scopus Google Scholar). The C. reinhardtii supercomplex has of Å Å Å overall and which compare with Å Å respectively, for the spinach LHCII·PSII supercomplex deduced from microscopy J. Morris E.P. B. M. Barber J. Nat. Struct. Biol. PubMed Scopus Google Scholar). The the of and is and and it is and 100 These are very similar to those of spinach, to be Å for both and with and and J. Morris E.P. B. M. Barber J. Nat. Struct. Biol. PubMed Scopus Google Scholar). The 3D structure of the S. elongatus core low to Å is in and is also a with protrusions are on the surface side which we to the OEC proteins. organization from that for C. reinhardtii and spinach. the to PsbP and PsbQ proteins in spinach and C. reinhardtii are and are replaced by a less protein mass In the region attributed to the PsbO protein is more comparable with the in the supercomplex structure. The complex has overall of Å Å Å overall and and the and is and and the are and a and projection maps of the region of C. reinhardtii and spinach the latter from J. Morris E.P. B. M. Barber J. Nat. Struct. Biol. PubMed Scopus Google the that were obtained different stained at for C. with for are similar in shape and a projection of the region of S. core with that of the spinach core complex also obtained from negatively stained and M. on Electron Scholar). The the core complexes of S. elongatus and spinach is more in where the two projection maps of the have been in is the positioning of the major transmembrane helices derived from electron crystallography on the spinach dimeric core complex B. Morris E.P. Barber J. Nat. Struct. Biol. 1999; 6: 560-564Crossref PubMed Scopus (116) Google Scholar). that the latter was obtained from microscopy and at a resolution of to the into the low resolution core maps is and consistent with J. Morris E.P. B. M. Barber J. Nat. Struct. Biol. PubMed Scopus Google Scholar, J. Nield J. Morris E.P. Hankamer B. Biochem. 1999; Scopus Google Scholar). in an using a PSII isolated from spinach E.J. Hankamer B. D. J. Nield J. Barber J. M. S. PubMed Scopus Google Scholar), the core complex is located in the supercomplex that the subunits are to the region (18Hankamer B. Nield J. Zheleva D. Boekema E.J. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (173) Google Scholar). In we have the projection maps of the core of S. elongatus and spinach into the projection of C. reinhardtii from The relative positioning of these negatively stained is on the structural model derived from spinach J. Nield J. Morris E.P. Hankamer B. Biochem. 1999; Scopus Google Scholar). can be the core complexes are into reinhardtii supercomplex The of the core projection into the C. reinhardtii structure also for the positioning of the transmembrane helices within the supercomplex and a to the relative location of the algal OEC proteins. b, are projection maps of the protrusions attributed to the OEC proteins of C. S. and spinach J. Morris E.P. B. M. Barber J. Nat. Struct. Biol. PubMed Scopus Google Scholar), These have been obtained by the of the complete 3D thus the masses of the OEC proteins. The of the organization of these proteins for C. reinhardtii and spinach is where are attributed to the PsbO protein and to the PsbP and PsbQ proteins of the The organization of the OEC proteins of S. elongatus is different The attributed to the PsbO protein and the less region seems to be a also by M. Boekema E.J. Eur. J. Biochem. 1999; PubMed Scopus Google Scholar). By the model for the positioning of the underlying transmembrane helices in this it is possible to a more complete of the organization of the OEC proteins for the In and the transmembrane helices have been on with 3D maps of the OEC with to in b, and It has previously been concluded from the microscopy and single-particle analysis of the spinach LHCII·PSII supercomplex that the PsbO protein was located the side of the whereas the region for the PsbP and PsbQ was over the lumenal surface of the N-terminal region of the D1 protein and the two transmembrane helices in the region J. Morris E.P. B. M. Barber J. Nat. Struct. Biol. PubMed Scopus Google Scholar). These relative positions are for the spinach model in but are also for C. reinhardtii The with S. elongatus is similar in that the density is located the side of the complex and is likely to be the protein. the other the of the density is less than the region in spinach and C. reinhardtii and to be continuous with the Nevertheless, this density also the lumenal surface of the N-terminal of the cyanobacterial D1 protein also incorporating the region by the two helices of and in that a to the algal and higher and also that of the to the PsbO is all OEC 3D are on of the the underlying transmembrane helices we report the 3D structural models of a supercomplex isolated from the green alga Chlamydomonas reinhardtii and of a core complex isolated from the thermophilic elongatus. both structures have been from data using negatively stained preparations and are to 30-Å some and to be made about the structure of PSII. The that a particle very similar to that isolated from spinach in C. reinhardtii suggests that the LHCII·PSII supercomplex be a in on with spinach, the LHCII·PSII supercomplex about particle and, 100 reaction center, that each supercomplex is dimeric (18Hankamer B. Nield J. Zheleva D. Boekema E.J. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (173) Google Scholar). of these are chlorophyll b, whereas the are The chlorophyll is consistent with each supercomplex of LHCII and a each of and reaction center (18Hankamer B. Nield J. Zheleva D. Boekema E.J. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (173) Google Scholar). The chlorophyll proteins are located the outer region of the whereas the region is composed of a dimeric core the major subunits D1, D2, and CP47, as as a of proteins J. Nield J. Morris E.P. Hankamer B. Biochem. 1999; Scopus Google Scholar, J. Biochim. Biophys. Acta. 1998; PubMed Scopus Google Scholar). The OEC proteins bind to the lumenal surface of the core. The core of S. elongatus has a structure similar to that of the core of spinach with the of the OEC proteins. was E.J. Hankamer B. D. J. Nield J. Barber J. M. S. PubMed Scopus Google Scholar), but is by of the of the 3D structures the structural features of the PSII core seems to be higher and cyanobacterial of the structural the different photosynthetic it is possible to a model of transmembrane organization obtained by electron crystallography for spinach PSII (5Rhee K.-H. Morris E.P. Zheleva D. Hankamer B. Kühlbrandt W. Barber J. Nature. 1997; 389: 522-526Crossref Scopus (127) Google Scholar, 6Rhee K.-H. Morris E.P. Barber J. Kühlbrandt W. Nature. 1998; 396: 283-286Crossref PubMed Scopus (284) Google Scholar). a a of the and in the location and of the OEC proteins in the different systems can be that in all the PsbO protein is positioned on the lumenal surface the side of the core. In helices and of CP47 are by the PsbO and the large that be in this protein. The of the five helices to CP47 to those of the D2 protein is and on J. Nield J. Morris E.P. Hankamer B. Biochem. 1999; Scopus Google Scholar). this is correct the PsbP and PsbQ proteins are located on the lumenal surface at the N-terminal of the D1 protein and also two helices of two low molecular weight proteins. is also for the of in the structure of S. which we to PsbU and PsbV c the molecular mass of PsbU and PsbV is it is that to be in size and have a with the surface of the core with the PsbP/PsbQ proteins, which have a molecular mass of about kDa. the low resolution of the it is of that the of the center of the mass attributed to the relative to the underlying transmembrane is for all structural it seems that the PsbP/PsbQ proteins of spinach C. reinhardtii and the of S. elongatus are located over the of the D1 protein and the two helices of located at the of the D1 protein. from resolution using complexes and structures that the and PsbU extrinsic proteins are attached to the of the and PsbV c 550) extrinsic proteins, respectively, for plants and cyanobacterial systems in with binding and B. Barber J. in The Scholar, Ikeuchi M. Inoue Y. FEBS Lett. 1994; PubMed Scopus Google Scholar). However, single-particle analyses that the and PsbV (Cytc 550) extrinsic proteins are located to the protein. is also consistent with B. Barber J. in The Scholar, Ikeuchi M. Inoue Y. FEBS Lett. 1994; PubMed Scopus Google Scholar, B. Res. Scopus Google Scholar), which that the protein the binding of the proteins, but its presence is for to the lumenal surface. In structural derived from single-particle that the to the and proteins are in and that this be more in to the lumenal for in and of the and Morris and Hankamer for The mutant was by Nixon and to we are