Annu. Rev. Cell Bioi. 1991. 7: 1-23 Copyright © 1991 by Ann ual Reviews In c. All rights reserved
ANNUAL REVIEWS
Further
Quick links to online content
STRUCTURES OF BACTERIAL PHOTOSYNTHETIC REACTION
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
CENTERS Johann Deisenhofer Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center,Dallas, Texas 75 235
Hartmut Michel Max- Planck-Institut fUr Biophysik, Abteilung Molekulare Membranbiochemie, Frankfurt/Main, Germany KEY WORDS:
Rhodopseudomonas viridis, membrane protein structure, X-ray crystallography, photosynthetic light reactions, electron transfer
CONTENTS PERSPECTIVES AND OVERVIEW
. . .. . . . . . .. . . . . . . . . . ... . . . . . . . . . . . . . .. . . . ... . . . . . . . .. . . . . . . . . .. . . ....... .. . . . . . . . . . .. .. .
1
CRYSTALLIZATION AND X-RAY STRUCTURE ANALYSIS OF RCS...........................................
3
The RC from Rhodopseudomonas viridis . . . . ........ . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . .. The RC from Rhodobacter sphaeroides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 10
IMPLICATIONS................................................................................................................
Path way s and Mechan ism o f Electron Tran sfer ... . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . .. . . . . . . . .. . . ..... R C Mutants ......... . .................................... . ....................................... ... .................... Photo sy stem II R C: S im ilar ity to R Cs of Purp le Bacter ia . . .. . .... . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . Membrane Protein Structure .. . . . . . . . . . . . . . . . . . . .. . . . . . . . .... . . . . . . .. . .. . . . . . . .. . . . . . . . . .. . . . .. . . ... . . . . . . . . ... . .... . . . .. ..... . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . ... . . . . . . . . . . .. . .... . .. . . . . . . . . . . . . . . . .. ... . . . . . . . ... .. . . Cr yst al logr aph y. . . . . . . . .. . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . .... . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . ... . . . . . Electron M icro scopy. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . .. . ... . . . . . . . . . . . .
PROSPECTS
11 11 13 14 15 16 16
17
PERSPECTIVES AND OVERVIEW Photosynthetic reaction centers (Res) are membrane-spanning complexes
of polypeptide chains and cofactors that catalyze the first steps in the conversion of light energy to chemical energy during photosynthesis. 1
0743-4634/ 9 1/ 1 1 15-000 1$0 2.00
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
2
DEISENHOFER & MICHEL
Absorption of the energy of a photon in the R C is followed by rapid, efficient electron transfer from a primary donor along a chain of acceptors leading across the photosynthetic membrane. In R Cs from photosynthetic purple bacteria,or from photosystem I I of green plants and cyanobacteria, the end of this chain is a quinone; during two functional cycles of the R C this quinone accepts two electrons and two protons and dissociates as a quinol from the R C. The end of the electron-transfer chain in photosystem I is an iron-sulphur center; this center reduces the small, water-soluble, electron-transport protein ferredoxin ( Golbeck 1 987). Photosynthetic purple bacteria were the fri st were discovered (see Duysens 1 98 9); their relative simplicity made them logical targets for studying photosynthetic light reactions. Reed & Clayton ( 1968) reported the first preparation Rhodopseudomonas (Rps.) sphaeorides (later renamed Rhodobacter (Rb.) �phaeroides), which has been one of the most popular organisms in photo synthesis research. In spite of rapid progress with photosystems of green plants and cyanobacteria, the R Cs of photosynthetic purple bacteria are still the best known mediators of primary photosynthetic processes. In particular, high-resolution three-dimensional structures of only two R Cs have been determined, both are from purple bacteria. R Cs of photosynthetic purple bacteria consist of at least three protein subunits called L (light), M (medium), and H (heavy), for the apparent molecular weights determined with SDS gel electrophoresis ( Feher & Okamura 1978). Subunits Land M have very hydrophobic amino acid se quences,in which five ( Williams et al 1 983, 1984; Youvan et al 1 984; -Michel et al 1 988); subunit H has a single membrane spanning segment near its amino terminus ( Youvan et al 1 984; Michel et al 1 985; Williams et al 1986). The subunits Land M bind bacteriochlorophylls ( B Chl), bacterio pheophytins ( BPh) , quinones, a ferrous iron ion, and a carotenoid as cofactors. These cofactors come in a variety of slightly different forms, e.g. B Chl and BPh of type a or b, in different species. Amino acid se quence homologies between subunits from four different species range from 60 to 78% for subunit L, and from 50 to 77% for subunit M ( Belanger et al 1 988). The currently known se quences of three H-subunits show homologies between 38 and 6 4% ( Williams et al 1986). A major difference between R Cs from different species of photosynthetic purple bacteria is the presence or absence of a four-heme c-type cyto chrome as the fourth protein subunit. Such a subunit is par t of the R C from e.g. Rps. viridis and Thiocapsa pfennigii; R Cs of other bacteria, e.g. Rb. sphaeroides, Rb. capsu!atus, and Rhodospirillum rubrum, consist of only the three protein subunits L, M, and H.
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
REACTION CENTER STRUCTURES
3
In this review, we focus on X-ray crystallographic studies on RCs from purple bacteria and briefly describe their structures using the R C from Rps. viridis as an example. We then discuss implications of the structures for RC function, relations to photosystem I I, and aspects of membrane protein structure. Finally, we mention new developments in structural studies of other photosynthetic membrane protein complexes. Recent reviews discuss various aspects of R Cs: structure and function (Parson 198 7; Budil et a 1 1987; Feher et aI 1 98 9); spectroscopy and elec tron transfer ( Friesner & Won 1989; Boxer 1 990); site-specific muta genesis ( Coleman & Youvan 1990); primary photochemistry (Kirmaier & Holten 1987; Budil et al 1 987); and membrane protein structure ( Rees et al 198 9b; Michel & Deisenhofer 1 990). Useful collections of articles on these topics can also be found in conference proceedings (Michel Beyerle 198 5; Breton & Vermeglio 1988 ). Articles describing aspects of membrane protein crystallization are collected e.g. in a recent monograph (Michel 1 990).
CRYSTALLIZATION AND X-RAY STRUCTURE ANALYSIS OF RCs To date, structural information to atomic detail on RCs has been ob tained only by using X-ray crystallography. The resolution of a crystal structure analysis is determined by the limit, usually expressed as the minimum Bragg spacing, to which X-ray reflections can be measured; this limit is in turn determined by the degree of long-range order in the crystal. Under most circumstances a resolution between 3.5 and 3.0 A is sufficient to construct a reasonably accurate atomic model of a molecule. A prere quisite for X-ray crystallography is the formation of well-ordered three-dimensional crystals. Crystallization is in many cases the highest hurdle on the way towards high-resolution structures of macromolecules. Until about 1980, three-dimensional crystallization of integral membrane proteins was widely considered impossible. Then three-dimensional crys tals were obtained for bacteriorhodopsin (Michel & Oesterhelt 1980) and matrix porin from Escherichia coli ( Garavito & Rosenbusch 1980), using the detergent octylglucoside to form water-solubleprotein-detergent com plexes that could be treated like soluble proteins in vapor diffusion experi ments in which ammonium sulphate or polyethylene glycol served as precipitants. These experiments were soon followed by the crystallization (Michel 1982) and X-ray structure analysis (Deisenhofer et a 1 1984) of the R C from Rps. viridis.
4
DEISENHOFER & MICHEL
The RC from
Rhodopseudomonas viridis
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
COMPOSITION The R C from the purple bacterium Rps. viridis is a complex of 4 protein subunits and 14 cofactors. The protein subunits are, in order of decreasing size, cytochrome [336 amino acids (aa)]; subunit M (323 aa); subunit L ( 273 aa); and subunit H ( 258 aa). The complete amino acid se quences of these subunits were derived from the se quences of the genes cod ing for them (Michel et al 1985, 1986; Weyer et al 1987a). The cofactors are four heme groups, covalently linked to the cytochrome subunit,four B Chl-b, two BPh-b, a mena quinone-9 and a ubi quinone-9, a ferrous iron ion, and the carotenoid dihydroneurosporene ( Thornber et a 1 1980; Gast et aI 1985).
CRYSTALS
Well-ordered three-dimensional crystals of the R C from Rps. obtained from R Cs solubilized with the detergent N,N-di methyl-dodecylamine- N-oxide (LD A O); the small amphiphile heptane1,2,3-triol was an essential additive, and ammonium sulphate was the precipitant in a vapor diffusion experiment (Michel 198 2, 1983). The crystals have the symmetry of space group P43 21 2. The unit cell constants are a b 223.5 A, c 1 13.6 A, and there are 8 R C molecules per unit cell (Michel 198 2;Deisenhofer et al 1984). The crystals diffract X-rays to 2.3 A resolution; at temperatures around 4°C they are rather insensitive to radiation damage, so that a substantial portion of a diffraction data set can be collected from one crystal (Deisenhofer et al 1984). STRUCTURE ANALYSIS The structure of the R C crystals was initially deter mined at 3.0 A resolution using the method of multiple isomorphous replacement with heavy atom compounds. An atomic model of the R C was built for the cofactors (Deisenhofer et a 1 1984) and the protein subunits (Deisenhofer et al 1985). The combination of se quence information and crystallographic data allowed the description of protein-cofactor inter actions (Michel et aI 1986).Crystallographic refinement (J. Deisenhofer, O. Epp, I. Sinning, H. Michel, in preparation) led to the current R C model consisting of 10,288 atoms, including 20 1 bound water molecules, 7 bound anions, and 1 firmly hofer & Michel 1988, 1989a,b). The upper limit for the average error in atomic coordinates (Luzzati 195 2) was estimated as 0.266 A (Deisenhofer &MicheI 1989a,b). In a low-resolution neutron diffraction study, the dis tribution of the detergent in the crystal could be determined ( Roth et aI 1989). RC MODEL Figure 1 shows an overall view of the model of the R C from Rps. viridis. In its longest dimension, the complex measures 130 A;the maxi mum width perpendicular to that direction is 70 A. The closely asso ciated subunits L and M, together with the bound B Chl, P Bh, quinones, nonheme iron, and carotenoid, form the central part of the R C. The most viridis were
=
=
=
�
�
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
REACTION CENTER STRUCTURES
Figure 1
5
Overall view of the RC from Rps. v ir idis. The polypeptide chains of the protein
subunits are represented by ribbons; cofactors are drawn as wire models. The membrane with polar head groups and hydrophobic core is shown schematically. The H-subunit pro trudes into the cytoplasm, the cytochrome into the periplasm.
prominent structural features of each of the central subunits are five long
hydrophobic helices that are assumed to span the bacterial membrane. This assumption is supported by experimental evidence (Roth et aI 1 989),by pro perties of the model (Deisenhofer & Michel 1 98 9b), and by functional considerations. The polypeptide backbones of subunits L and M and the attached cofactors display a high degree of local twofold symmetry;the local twofold symmetry axis is oriented perpendicular to the membrane plane ( Breton 1 985). On either side of the membrane-spanning region of the L-M complex, a peripheral subunit is attached: the cytochrome with its four bound heme groups is atthe periplasmic side,and the globular domain
6
DEISENHOFER & MICHEL
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
of the H-subunit is at the cytoplasmi c side of the membrane. The H-subunit contributes a single membrane-spanning helix. Neither the cyto chrome nor the H-subunit obeys the lo cal symmetry found in the central part of the R C; the cyto chrome has internal lo cal symmetry of its own. Figure 2 shows s chemati c drawings of the polypeptide chain-folding of the rea ction center subunits Land M; the stru ctural similarity between these subunits is obvious. Stru cturally similar segments in the L and M subunits in clude the transmembrane heli ces (labeled A, E, C, D, E) and a large fra ction of the conne ctions. In total, 2 16 IX carbons from the M
(a)
(b)
MBO
Figure 2
(stereo pair): L-subunit (a) and M-subunit (b) of the
MBO
RC
from Rps. viridis in
ribbon representation. The transmembrane helices in each subunit are labeled A, B, C, D, andE.
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
REACTION CENTER STRUCTURES
7
subunit can be superimposed onto corresponding \1. carbons of the L subunit by a rotation of almost exa ctly 1800to a root mean s quare (rms) deviation of 1.22 A. The transmembrane heli ces are between 2 1 and 28 residues long;the conne cting segments range in length from 4 to 34 residues and contain shorter heli ces. The M subunit is 50 residues longer than the L subunit. Both se quen ce alignments (Mi chel et al 1986) and stru ctural alignments (Deisenhofer et al 1985; Deisenhofer & Mi chel 1989b) show that the se quen ce insertions in M are lo cated on either side of the trans membrane heli ces (20 at the NH2 terminus, 7 in the conne ction between transmembrane heli ces M A and M B, 7 in the conne ction between MD and M E, and 16 at the C O OH terminus). Through these insertions, the M subunit dominates the conta cts of the L-M complex to the peripheral subunits. Also, the insertions help create conta ct surfa ces for the peripheral subunits that deviate signifi cantly from twofold symmetry. The insertion between transmembrane heli ces MD and M E, containing another small helix,is of importan ce for binding of the nonheme iron and for the different conformations of the quinone-binding sites in L and M. The H-subunit, shown in Figure 3, can be divided into three stru ctural regions with different chara cteristi cs. The first region (residues H I to H39) contains the only transmembrane helix of subunit H (residues H12 to H35). Residue HI is formyl-methionine; the end of the region, near the cytoplasmi c end of the transmembrane helix, has seven conse cutive charged residues (H33 to H39). The se cond stru ctural region of the H
Figure 3
(stereo pair): The H-subunit of the RC from Rps. viridis in ribbon representation.
Residues H47 to H53 are disordered in the crystal and not shown.
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
8
DEISENHOFER & MICHEL
chain (residues H 40 to H10 4) mostly follows the surfa ce of the L-M complex, apparently deriving structural stability from that contact. This surface region contains a short helix and two two-stranded antiparallel {3sheets. Residues H47 to H53 la ck conta ct with the rest of the R C stru cture and are disordered in the crystal so that no signifi be found for them. The third stru ctural segment of the H-subunit,starting at about H 105; forms a globular domain. This domain contains an extended system of parallel and antiparallel {3-sheets (between residues H l 34 and H 203) and an a-helix (residues H 232 to H 248). The cytochrome is the largest subunit in the rea ction center complex ( Weyer et al 1987a). Its stru cture can be divided into an NH2-terminal segment, two pairs ofheme-binding segments, and a segment connecting the two pairs. Each heme-binding segment consists of a helix with an average length of 17 residues followed by a turn and the Cys- X- Y- Cys His se quence, typical for c-type cytochromes. The hemes are connected to the cysteine residues via thioether linkages. The heme planes are parallel to the helix axes. The sixth ligand to the heme iron is a methionine residue found within the helix in three of the four cases. Histidine C 1 24, located in a different part of the structure, serves as a sixth ligand for the iron of heme 4. The two pairs ofheme-binding segments,containing hemes 1 and 2, and 3 and 4, respe ctively, are related by lo cal twofold symmetry. Sixty five residues ofeach pair obey this local symmetry with an rms deviation between corresponding a carbon atoms of 0.93 A. The local symmetry of the cytochrome is not related to the local symmetry between the subunits L and M. The last four residues ofthe cytochrome subunit, C333 to C336, are disordered. Also disordered is the lipid moiety bound to the NH2 terminal cysteine residue ( Weyer et al 1987b). Figure 4 shows arrangement and nomenclature ofthe cofactors associ ated with the protein subunits L and M, excluding the carotenoid, the phytyl side chains of B Chl-b and BPh-b,and the isoprenoid side chains of quinones forclarity. A closely asso ciated pair of B Chl-b, the spe cial pair, resides at the origin oftwo branches ofcofactors, each of which consists of another B Chl-b (the accessory B Chl-b), a B Ph-b, and a quinone. The nonheme iron sits between the quinones and is bound to five residues, four histidines (Ll90, L 230, M 2 17, M 264), and one glutamic acid (M 232) (Deisenhofer et al 1985; Michel et al 1986). The Mg2+ ions ofthe B Chl-b are five-coordinated the subunits L and M acting as ligands (L lS3-+ BA,L l73-+DL, M 180-+ BB, M 200 -+DM). DL, DM,
ANNUAL REVIEWS
Further
Quick links to online content
STRUCTURES OF BACTERIAL PHOTOSYNTHETIC REACTION
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
CENTERS Johann Deisenhofer Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center,Dallas, Texas 75 235
Hartmut Michel Max- Planck-Institut fUr Biophysik, Abteilung Molekulare Membranbiochemie, Frankfurt/Main, Germany KEY WORDS:
Rhodopseudomonas viridis, membrane protein structure, X-ray crystallography, photosynthetic light reactions, electron transfer
CONTENTS PERSPECTIVES AND OVERVIEW
. . .. . . . . . .. . . . . . . . . . ... . . . . . . . . . . . . . .. . . . ... . . . . . . . .. . . . . . . . . .. . . ....... .. . . . . . . . . . .. .. .
1
CRYSTALLIZATION AND X-RAY STRUCTURE ANALYSIS OF RCS...........................................
3
The RC from Rhodopseudomonas viridis . . . . ........ . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . .. The RC from Rhodobacter sphaeroides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 10
IMPLICATIONS................................................................................................................
Path way s and Mechan ism o f Electron Tran sfer ... . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . .. . . . . . . . .. . . ..... R C Mutants ......... . .................................... . ....................................... ... .................... Photo sy stem II R C: S im ilar ity to R Cs of Purp le Bacter ia . . .. . .... . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . Membrane Protein Structure .. . . . . . . . . . . . . . . . . . . .. . . . . . . . .... . . . . . . .. . .. . . . . . . .. . . . . . . . . .. . . . .. . . ... . . . . . . . . ... . .... . . . .. ..... . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . ... . . . . . . . . . . .. . .... . .. . . . . . . . . . . . . . . . .. ... . . . . . . . ... .. . . Cr yst al logr aph y. . . . . . . . .. . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . .... . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . ... . . . . . Electron M icro scopy. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . .. . ... . . . . . . . . . . . .
PROSPECTS
11 11 13 14 15 16 16
17
PERSPECTIVES AND OVERVIEW Photosynthetic reaction centers (Res) are membrane-spanning complexes
of polypeptide chains and cofactors that catalyze the first steps in the conversion of light energy to chemical energy during photosynthesis. 1
0743-4634/ 9 1/ 1 1 15-000 1$0 2.00
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
2
DEISENHOFER & MICHEL
Absorption of the energy of a photon in the R C is followed by rapid, efficient electron transfer from a primary donor along a chain of acceptors leading across the photosynthetic membrane. In R Cs from photosynthetic purple bacteria,or from photosystem I I of green plants and cyanobacteria, the end of this chain is a quinone; during two functional cycles of the R C this quinone accepts two electrons and two protons and dissociates as a quinol from the R C. The end of the electron-transfer chain in photosystem I is an iron-sulphur center; this center reduces the small, water-soluble, electron-transport protein ferredoxin ( Golbeck 1 987). Photosynthetic purple bacteria were the fri st were discovered (see Duysens 1 98 9); their relative simplicity made them logical targets for studying photosynthetic light reactions. Reed & Clayton ( 1968) reported the first preparation Rhodopseudomonas (Rps.) sphaeorides (later renamed Rhodobacter (Rb.) �phaeroides), which has been one of the most popular organisms in photo synthesis research. In spite of rapid progress with photosystems of green plants and cyanobacteria, the R Cs of photosynthetic purple bacteria are still the best known mediators of primary photosynthetic processes. In particular, high-resolution three-dimensional structures of only two R Cs have been determined, both are from purple bacteria. R Cs of photosynthetic purple bacteria consist of at least three protein subunits called L (light), M (medium), and H (heavy), for the apparent molecular weights determined with SDS gel electrophoresis ( Feher & Okamura 1978). Subunits Land M have very hydrophobic amino acid se quences,in which five ( Williams et al 1 983, 1984; Youvan et al 1 984; -Michel et al 1 988); subunit H has a single membrane spanning segment near its amino terminus ( Youvan et al 1 984; Michel et al 1 985; Williams et al 1986). The subunits Land M bind bacteriochlorophylls ( B Chl), bacterio pheophytins ( BPh) , quinones, a ferrous iron ion, and a carotenoid as cofactors. These cofactors come in a variety of slightly different forms, e.g. B Chl and BPh of type a or b, in different species. Amino acid se quence homologies between subunits from four different species range from 60 to 78% for subunit L, and from 50 to 77% for subunit M ( Belanger et al 1 988). The currently known se quences of three H-subunits show homologies between 38 and 6 4% ( Williams et al 1986). A major difference between R Cs from different species of photosynthetic purple bacteria is the presence or absence of a four-heme c-type cyto chrome as the fourth protein subunit. Such a subunit is par t of the R C from e.g. Rps. viridis and Thiocapsa pfennigii; R Cs of other bacteria, e.g. Rb. sphaeroides, Rb. capsu!atus, and Rhodospirillum rubrum, consist of only the three protein subunits L, M, and H.
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
REACTION CENTER STRUCTURES
3
In this review, we focus on X-ray crystallographic studies on RCs from purple bacteria and briefly describe their structures using the R C from Rps. viridis as an example. We then discuss implications of the structures for RC function, relations to photosystem I I, and aspects of membrane protein structure. Finally, we mention new developments in structural studies of other photosynthetic membrane protein complexes. Recent reviews discuss various aspects of R Cs: structure and function (Parson 198 7; Budil et a 1 1987; Feher et aI 1 98 9); spectroscopy and elec tron transfer ( Friesner & Won 1989; Boxer 1 990); site-specific muta genesis ( Coleman & Youvan 1990); primary photochemistry (Kirmaier & Holten 1987; Budil et al 1 987); and membrane protein structure ( Rees et al 198 9b; Michel & Deisenhofer 1 990). Useful collections of articles on these topics can also be found in conference proceedings (Michel Beyerle 198 5; Breton & Vermeglio 1988 ). Articles describing aspects of membrane protein crystallization are collected e.g. in a recent monograph (Michel 1 990).
CRYSTALLIZATION AND X-RAY STRUCTURE ANALYSIS OF RCs To date, structural information to atomic detail on RCs has been ob tained only by using X-ray crystallography. The resolution of a crystal structure analysis is determined by the limit, usually expressed as the minimum Bragg spacing, to which X-ray reflections can be measured; this limit is in turn determined by the degree of long-range order in the crystal. Under most circumstances a resolution between 3.5 and 3.0 A is sufficient to construct a reasonably accurate atomic model of a molecule. A prere quisite for X-ray crystallography is the formation of well-ordered three-dimensional crystals. Crystallization is in many cases the highest hurdle on the way towards high-resolution structures of macromolecules. Until about 1980, three-dimensional crystallization of integral membrane proteins was widely considered impossible. Then three-dimensional crys tals were obtained for bacteriorhodopsin (Michel & Oesterhelt 1980) and matrix porin from Escherichia coli ( Garavito & Rosenbusch 1980), using the detergent octylglucoside to form water-solubleprotein-detergent com plexes that could be treated like soluble proteins in vapor diffusion experi ments in which ammonium sulphate or polyethylene glycol served as precipitants. These experiments were soon followed by the crystallization (Michel 1982) and X-ray structure analysis (Deisenhofer et a 1 1984) of the R C from Rps. viridis.
4
DEISENHOFER & MICHEL
The RC from
Rhodopseudomonas viridis
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
COMPOSITION The R C from the purple bacterium Rps. viridis is a complex of 4 protein subunits and 14 cofactors. The protein subunits are, in order of decreasing size, cytochrome [336 amino acids (aa)]; subunit M (323 aa); subunit L ( 273 aa); and subunit H ( 258 aa). The complete amino acid se quences of these subunits were derived from the se quences of the genes cod ing for them (Michel et al 1985, 1986; Weyer et al 1987a). The cofactors are four heme groups, covalently linked to the cytochrome subunit,four B Chl-b, two BPh-b, a mena quinone-9 and a ubi quinone-9, a ferrous iron ion, and the carotenoid dihydroneurosporene ( Thornber et a 1 1980; Gast et aI 1985).
CRYSTALS
Well-ordered three-dimensional crystals of the R C from Rps. obtained from R Cs solubilized with the detergent N,N-di methyl-dodecylamine- N-oxide (LD A O); the small amphiphile heptane1,2,3-triol was an essential additive, and ammonium sulphate was the precipitant in a vapor diffusion experiment (Michel 198 2, 1983). The crystals have the symmetry of space group P43 21 2. The unit cell constants are a b 223.5 A, c 1 13.6 A, and there are 8 R C molecules per unit cell (Michel 198 2;Deisenhofer et al 1984). The crystals diffract X-rays to 2.3 A resolution; at temperatures around 4°C they are rather insensitive to radiation damage, so that a substantial portion of a diffraction data set can be collected from one crystal (Deisenhofer et al 1984). STRUCTURE ANALYSIS The structure of the R C crystals was initially deter mined at 3.0 A resolution using the method of multiple isomorphous replacement with heavy atom compounds. An atomic model of the R C was built for the cofactors (Deisenhofer et a 1 1984) and the protein subunits (Deisenhofer et al 1985). The combination of se quence information and crystallographic data allowed the description of protein-cofactor inter actions (Michel et aI 1986).Crystallographic refinement (J. Deisenhofer, O. Epp, I. Sinning, H. Michel, in preparation) led to the current R C model consisting of 10,288 atoms, including 20 1 bound water molecules, 7 bound anions, and 1 firmly hofer & Michel 1988, 1989a,b). The upper limit for the average error in atomic coordinates (Luzzati 195 2) was estimated as 0.266 A (Deisenhofer &MicheI 1989a,b). In a low-resolution neutron diffraction study, the dis tribution of the detergent in the crystal could be determined ( Roth et aI 1989). RC MODEL Figure 1 shows an overall view of the model of the R C from Rps. viridis. In its longest dimension, the complex measures 130 A;the maxi mum width perpendicular to that direction is 70 A. The closely asso ciated subunits L and M, together with the bound B Chl, P Bh, quinones, nonheme iron, and carotenoid, form the central part of the R C. The most viridis were
=
=
=
�
�
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
REACTION CENTER STRUCTURES
Figure 1
5
Overall view of the RC from Rps. v ir idis. The polypeptide chains of the protein
subunits are represented by ribbons; cofactors are drawn as wire models. The membrane with polar head groups and hydrophobic core is shown schematically. The H-subunit pro trudes into the cytoplasm, the cytochrome into the periplasm.
prominent structural features of each of the central subunits are five long
hydrophobic helices that are assumed to span the bacterial membrane. This assumption is supported by experimental evidence (Roth et aI 1 989),by pro perties of the model (Deisenhofer & Michel 1 98 9b), and by functional considerations. The polypeptide backbones of subunits L and M and the attached cofactors display a high degree of local twofold symmetry;the local twofold symmetry axis is oriented perpendicular to the membrane plane ( Breton 1 985). On either side of the membrane-spanning region of the L-M complex, a peripheral subunit is attached: the cytochrome with its four bound heme groups is atthe periplasmic side,and the globular domain
6
DEISENHOFER & MICHEL
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
of the H-subunit is at the cytoplasmi c side of the membrane. The H-subunit contributes a single membrane-spanning helix. Neither the cyto chrome nor the H-subunit obeys the lo cal symmetry found in the central part of the R C; the cyto chrome has internal lo cal symmetry of its own. Figure 2 shows s chemati c drawings of the polypeptide chain-folding of the rea ction center subunits Land M; the stru ctural similarity between these subunits is obvious. Stru cturally similar segments in the L and M subunits in clude the transmembrane heli ces (labeled A, E, C, D, E) and a large fra ction of the conne ctions. In total, 2 16 IX carbons from the M
(a)
(b)
MBO
Figure 2
(stereo pair): L-subunit (a) and M-subunit (b) of the
MBO
RC
from Rps. viridis in
ribbon representation. The transmembrane helices in each subunit are labeled A, B, C, D, andE.
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
REACTION CENTER STRUCTURES
7
subunit can be superimposed onto corresponding \1. carbons of the L subunit by a rotation of almost exa ctly 1800to a root mean s quare (rms) deviation of 1.22 A. The transmembrane heli ces are between 2 1 and 28 residues long;the conne cting segments range in length from 4 to 34 residues and contain shorter heli ces. The M subunit is 50 residues longer than the L subunit. Both se quen ce alignments (Mi chel et al 1986) and stru ctural alignments (Deisenhofer et al 1985; Deisenhofer & Mi chel 1989b) show that the se quen ce insertions in M are lo cated on either side of the trans membrane heli ces (20 at the NH2 terminus, 7 in the conne ction between transmembrane heli ces M A and M B, 7 in the conne ction between MD and M E, and 16 at the C O OH terminus). Through these insertions, the M subunit dominates the conta cts of the L-M complex to the peripheral subunits. Also, the insertions help create conta ct surfa ces for the peripheral subunits that deviate signifi cantly from twofold symmetry. The insertion between transmembrane heli ces MD and M E, containing another small helix,is of importan ce for binding of the nonheme iron and for the different conformations of the quinone-binding sites in L and M. The H-subunit, shown in Figure 3, can be divided into three stru ctural regions with different chara cteristi cs. The first region (residues H I to H39) contains the only transmembrane helix of subunit H (residues H12 to H35). Residue HI is formyl-methionine; the end of the region, near the cytoplasmi c end of the transmembrane helix, has seven conse cutive charged residues (H33 to H39). The se cond stru ctural region of the H
Figure 3
(stereo pair): The H-subunit of the RC from Rps. viridis in ribbon representation.
Residues H47 to H53 are disordered in the crystal and not shown.
Annu. Rev. Cell. Biol. 1991.7:1-23. Downloaded from www.annualreviews.org by Grand Valley State University on 11/19/13. For personal use only.
8
DEISENHOFER & MICHEL
chain (residues H 40 to H10 4) mostly follows the surfa ce of the L-M complex, apparently deriving structural stability from that contact. This surface region contains a short helix and two two-stranded antiparallel {3sheets. Residues H47 to H53 la ck conta ct with the rest of the R C stru cture and are disordered in the crystal so that no signifi be found for them. The third stru ctural segment of the H-subunit,starting at about H 105; forms a globular domain. This domain contains an extended system of parallel and antiparallel {3-sheets (between residues H l 34 and H 203) and an a-helix (residues H 232 to H 248). The cytochrome is the largest subunit in the rea ction center complex ( Weyer et al 1987a). Its stru cture can be divided into an NH2-terminal segment, two pairs ofheme-binding segments, and a segment connecting the two pairs. Each heme-binding segment consists of a helix with an average length of 17 residues followed by a turn and the Cys- X- Y- Cys His se quence, typical for c-type cytochromes. The hemes are connected to the cysteine residues via thioether linkages. The heme planes are parallel to the helix axes. The sixth ligand to the heme iron is a methionine residue found within the helix in three of the four cases. Histidine C 1 24, located in a different part of the structure, serves as a sixth ligand for the iron of heme 4. The two pairs ofheme-binding segments,containing hemes 1 and 2, and 3 and 4, respe ctively, are related by lo cal twofold symmetry. Sixty five residues ofeach pair obey this local symmetry with an rms deviation between corresponding a carbon atoms of 0.93 A. The local symmetry of the cytochrome is not related to the local symmetry between the subunits L and M. The last four residues ofthe cytochrome subunit, C333 to C336, are disordered. Also disordered is the lipid moiety bound to the NH2 terminal cysteine residue ( Weyer et al 1987b). Figure 4 shows arrangement and nomenclature ofthe cofactors associ ated with the protein subunits L and M, excluding the carotenoid, the phytyl side chains of B Chl-b and BPh-b,and the isoprenoid side chains of quinones forclarity. A closely asso ciated pair of B Chl-b, the spe cial pair, resides at the origin oftwo branches ofcofactors, each of which consists of another B Chl-b (the accessory B Chl-b), a B Ph-b, and a quinone. The nonheme iron sits between the quinones and is bound to five residues, four histidines (Ll90, L 230, M 2 17, M 264), and one glutamic acid (M 232) (Deisenhofer et al 1985; Michel et al 1986). The Mg2+ ions ofthe B Chl-b are five-coordinated the subunits L and M acting as ligands (L lS3-+ BA,L l73-+DL, M 180-+ BB, M 200 -+DM). DL, DM,
