ANNUAL REVIEWS
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Annll, ReI). BioplJys. Biopl1rs. Chern. 1991. 20:247-{)6 Copyright © 1991 by Ann�al Reviews Inc. All rights resen'ed
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
HIGH-RESOLUTION STRUCTURES OF PHOTOSYNTHETIC REACTION CENTERS Johann Deisenhofer Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Hartmut Michel Max-Planck-Institut fiir Biophysik, Abteilung Molekulare Membranbiochemie, Frankfurt/Main, FRG KEY WORDS:
membrane protein structurc, RllOdospeudomonas viridis, Rhodo bacler sphaeroides, X-ray crystallography, photosynthetic purple bacteria
CONTENTS PERSPECTIVES AND OVERVIEW ..
248
CRYSTALLIZATION AND X-RAY STRUCTURE ANALYSIS OF Res ..
249
The RC from Rhodopseudomonas viridis .. The RC from Rhodobacter sphaeroides .....
250 257 258
IMPLlCATIONS..
PUlhway.i and Mechanism of Electron Transfer RC Mutants. .... ........... ....... .... Phnlosystem 1I RC: Similarity to RCs of Purple Bacteriu .... Membrane Protein Structure ........ . ..... .......
..
.
..
...
261
PROSPECTS ..
Crystallography .
.
258 259 260 260 261 263
.
Eleclrot1 Microscopy..
247 0883-9182/91/061 0--{)247$02.00
248
DEISENHOFER & MICHEL
PERSPECTIVES AND OVERVIEW Photosynthetic reaction centers (RCs) 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. Absorption of the energy of a photon in the RC is followed by rapid,
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
efficient electron transfer from a primary donor along a chain of acceptors leading across the photosynthetic membrane. [n RCs from photosynthetic purple bacteria, or from photosystem II of green plants and cyanobacteria , the end of this chain is a quinone that, after double reduction and pro tonation converts it into a quinol, dissociates from the RC. The end of the electron-transfer chain in photosystem I is an iron-sulphur center, and the small, water-soluble, electron-transport protein ferredoxin (47). Photosynthetic purple bacteria were the first organisms in which RCs were discovered (37); their relative simplicity made them logical targets for studying photosynthetic light reactions. Reed & Clayton (87) reported the first preparation of an RC, that from Rhodopseudomonas sphaeroides (later renamed Rhodobacter sphaeroides). In spite of rapid progress with photosystems of green plants and cyano bacteria, the RCs of photosynthetic purple bacteria are still the best known mediators of primary photosynthetic processes. In particular, only two high-resolution three-dimensional structures of RCs have been deter mined; both are from purple bacteria. RCs 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 (40). Subunits Land M have very hydrophobic amino acid sequences, in which five membrane-spanning segments can be distinguished (17, 72, 109, 110, 115); subunit H has a single membrane-spanning segment near its amino ter minus (73, 108, 115). The subunits Land M bind bacteriochlorophylls (BChl), bacteriopheophytins (BPh), quinones, a ferrous iron ion, and a carotenoid as cofactors. Species differ in the amino acid sequences of their protein subunits and in the types of cofactors bound to them. Amino acid sequence homologies between subunits from four different species range from 60 to 78% for subunit L, sequences of three H-subunits show homologies between 38 and 64% (108). BChl and BPh of type a (BChl-a, BPh-a) are found e.g. in RCs from
R. sphaeroides and Rhodobacter capsula/us; type b (BChl-b and BPh-b) are e.g. in Rhodopseudomonas viridis and Thiocapsa pfennigii (91). The quinones also can be of different types: R. sphaeroides has two ubiquinone-
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
REACTION CENTER STRUCTURES
249
10 molecules (39), whereas R. viridis has one menaquinone-9 and one ubiquinone-9 molecule (46). Various types of carotenoids are used in different species; for example in R. viridis, the major carotenoid species is 1,2-dihydroneurosporene (I. Sinning & H. Michel, unpublished data); in R. sphaeroides, it is spheroidene (21). Another major difference between RCs from different species of photo synthetic purple bacteria is the presence or absence of a four-heme c-type cytochrome as the fourth protein subunit. Such a subunit is part of the RC from e.g. R. viridis and T. pfennigii; RCs of other bacteria, e.g. R. sphaeroides, R. capsulatus, and Rhodospirillum rubrum, consist of only the three protein subunits L,M, and H. In this review, we focus on X-ray crystallographic studies on RCs from purple bacteria and briefly describe their structures using the RC from R. viridis as an example. We then discuss implications of the structures for RC function, relations to photosystem II, 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 RCs: structure and function (16, 39, 83), spectroscopy and electron transfer (12, 43), site-specific mutagenesis (23), primary photochemistry (16, 55), and membrane protein structure (69, 89). Useful collections of artieles on these topics can also be found in conference proceedings ( 1 5, 74). CRYSTALLIZATION AND X-RAY STRUCTURE ANALYSIS OF RCs
To date, high-resolution structural information on RCs has been obtained only by using X-ray crystallography. By "high resolution," we mean res olution sufficient to construct a reasonably accurate atomic model of a molecule; the resolution needed for a reliable model dcpcnds on thc quality and completeness of the diffraction data and on the accuracy of the phase information available; it usually lies between 3.5 and 3.0 A. A prerequisite 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 generally considered impossible. Then three-dimensional crystals were obtained for bacteriorhodopsin (7 1 ) and matrix porin from Escherichia coli (45) using the detergent octylglucoside to form water soluble protein-detergent complexes that could be treated like soluble proteins in vapor diffusion experiments in which ammonium sulphate or
250
DEISENHOFER & MICHEL
polyethylene glycol served as precipitants. These experiments were soon followed by the crystallization (65) and X-ray structure analysis (28) of the RC from R. viridis. The RC from Rhodopseudomonas viridis
The RC from the purple bacterium R. viridis is a complex of 4 protein subunits and 14 cofactors. The protein subunits are, in order of decreasing size, cytochrome [336 amino acids (a.a.)], subunit M (323 a.a.), subunit L (273 a.a.) and subunit H (258 a.a.); the complete amino acid sequences of these subunits were derived from the sequences of the genes coding for them (72, 73, 106). The cofactors are four heme groups, covalently linked to the cytochrome subunit, four BChl-b, two BPh-b, a menaquinone-9 and a ubiquinone-9, a ferrous iron ion, and the carotenoid dihydroneurosporene (46, 97).
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
COMPOSITION
Well-ordered three-dimensional crystals of the RC from R. were obtained from RCs solubilized with the detergent N,N-di methyl-dodecylamine-N-oxide (LDAO); the small amphiphile heptane1,2,3-triol was an essential additive, and ammonium sulphate was the precipitant in a vapor-diffusion experiment (65, 66). The crystals have the symmetry of space group P432]2. The unit cell constants are a b 223.5 A, c = 113.6 A, and 8 RC molecules per unit cell (28, 65). The crystals diffract X-rays to at least 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 (28). CRYSTALS
viridis
=
=
The structure of the RC crystals was initially determined at 3.0-A resolution using the method of multiple isomorphous replacement with heavy atom compounds (28). Small mercury compounds and uranyl nitrate were especially useful for that purpose. To preserve the high crystalline order during the soaking experiments, an additional purification step had to be introduced, and a special "soak buffer" had to be developed (28, 31). Phase information from five heavy atom derivatives enabled the calculation of an electron density map, which the method of solvent flattening (104) further improved. On the basis of this electron density map, an atomic model of the RC was built for the cofactors (28) and the protein subunits (29). The combination of sequence information and crystallographic data allowed the description of protein cofactor inter actions (70). Crystallographic refinement at 2.3-A resolution (1. Deisen hofer, O. Epp, I. Sinning, & H. Michel, in preparation) led to an atomic model consisting of 10,288 atoms, including e.g. 201 bound water molecules, 7 bound anions, and 1 firmly bound LDAO molecule (30-32). The upper limit for the average error in atomic coordinates (62) was X-RAY STRUCTURE ANALYSIS
REACTION CENTER STRUCTURES
251
estimated as 0.266 A (31, 32). The X-ray data did not allow distinction among carbon, nitrogen, and oxygen atoms. Orientations of side chains of amino acids such as asparagine, glutamine, histidine, and threonine, or of acetyl groups of BChl and BPh, were chosen to interact optimally with the environment. In a low-resolution neutron diffraction study, the distribution of the detergent in the crystal could be determined (90). Figure I shows an overall view of the model of the RC from 130 A; the maximum width perpendicular to that direction is 70 A. The closely associated subunits L and M, together with the bound Behl, BPh, quinones, nonheme iron, and carotenoid, form the central part of the RC. The most 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 (90), by properties of the model (31), and by functional considerations. The
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
RC MODEL
R. viridis. In its longest dimension, the complex measures
�
'"
Figure 1
(Stereo pair) Overall view of the RC from R. viridis. The polypeptide chains of
the protein subunits are represented by ribbons; co factors are drawn as wire models. The membrane plane is approximately perpendicular to the plane of projection. The approximate positions of the periplasmic (upper line) and the cytoplasmic (lower line) membrane surfaces are indicated.
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
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DEISENHOFER & MICHEL
polypeptide backbones of subunits L and M and the attached cofactors display a high degree of local two-fold symmetry; the local two-fold sym metry axis is oriented perpendicular to the membrane plane (13). 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 at the periplasmic side, and the globular domain of the H-subunit is at the cytoplasmic side of the membrane. The H-subunit contributes a single membrane-spanning helix. Neither the cytochrome nor the H-subunit obey the local symmetry found in the central part of the RC; the cytochrome has internal local symmetry of its own. Figure 2 shows schematic drawings of the polypeptide-chain folding of the reaction center subunits L and M; the structural similarity between these subunits is obvious. Structurally similar segments in the L and M subunits include the transmembrane helices (labeled A, B, C, D, E) and a large fraction of the connections. In total, 216 IJ. carbons from the M subunit can be superimposed onto corresponding IX carbons of the L subunit by a rotation of almost exactly 1800 to a root mean square (rms) deviation of 1.22 A. In addition to the transmembrane helices, which have lengths between 21 and 28 residues, the connecting segments contain shorter helices. The M subunit is 50 residues longer than the L subunit. Both sequence alignments (72) and structural alignments (29, 31) show that the sequence insertions in M reside on either side of the trans membrane helices (20 at the NH2 terminus, 7 in the connection between transmembrane helices MA and MB, 7 in the connection between MD and ME, and 16 at the COOH terminus). Through these insertions, the M subunit dominates the contacts of the L-M complex to the peripheral subunits. The insertion between transmembrane helices MD and ME, containing another small helix, is of importance 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 structural regions with different characteristics. The amino terminal segment, begin ning with formylmethionine (73), contains the only transmembrane helix of subunit H (residues H l 2 to H35). Near the end of the transmembrane helix, the sequence shows seven consecutive charged residues (H33 to H39). Residues H47 to HS3 are disordered in the crystal so that no significant electron density can be found for them. Following the dis ordered region, the H chain forms an extended structure along the surface of the L -M complex, apparently deriving structural stability from that contact. The surface region contains a short helix and two two-stranded antiparallel f3-sheets. The third structural segment of the H-subunit, start ing at about H l OS, forms a globular domain. This domain contains an
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
REACTION CENTER STRUCTURES
MBO
Figure 2
(Stereo
253
MBO
pairs) L-subunit (top) and M-subunit (bottom) of the RC from R. viridis
in ribbon representation. The transmembrane helices in each subunit are labeled A, B, C, D, and E.
extended system of parallel and antiparallel /I-sheets (between residues H134 and H203), and an a-helix (residues H232 to H248). The cytochrome is the largest subunit in the reaction center complex
(106).
Its structure, shown in Figure 4, consists of an NHz-terminal
segment, two pairs of heme-binding segments, and a segment connecting the two pairs. Each heme binding segment consists of a helix with an
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
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DEISENHOFER & MICHEL
Figure 3
(Stereo pair) The H-subunit of the RC from R. viridis in ribbon representation.
Residues H47 to H53 are disordered in the crystal and not shown.
average length of 17 residues followed by a turn and the Cys-X-y-Cys His sequence typical for c-type cytochromes. The hemes are connected to the cysteine residues via thioether linkages. This arrangement makes the heme planes parallel to the helix axes. The sixth ligand to the heme iron is a methionine residue within the helix in three of the four cases. Histidine C124, located in a different part of the structure, serves as a sixth ligand for the iron of heme 4. The two pairs of heme binding segments, containing hemes I and 2 and 3 and 4, respectively, are related by a local two-fold symmetry. Sixty-five residues of each pair obey this local symmetry with an rms deviation between corresponding IX carbon atoms of 0.93 A. The local symmetry of the cytochrome is not related to the central local sym metry. The last four residues, C333 to C336, are disordered. Also dis ordered is the lipid moiety bound to the NH2-terminal cysteine residue (107). Figure 5 shows arrangements and nomenclature of the cofactors associ ated with the protein subunits L and M, excluding phytyl side chains or isoprenoid side chains for clarity. A closely associated pair of BChl-bs, the special pair, resides at the origin of two branches of cofactors, each of which consists of another BChl-b (the accessory BChl-b), a BPh-b, and a quinone. The nonheme iron sits between the quinones and is bound to five
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
REACTION CENTER STRUCTURES
Figure 4
255
(Stereo pair) The cytochrome subunit of the RC from R. viridis. The polypeptide
chain is drawn as a ribbon; the hemes and the heme-binding cysteine residues are drawn as wire models. The view is approximately along the local two-fold symmetry axis of the cytochrome. The hemes are numbered 1,2,4,3 (top to bottom; numbers not shown).
Figure 5
(Stereo pair) Model of the co factors associated with subunits L and M in the RC
from R. viridis. The molecules are labeled according to the nomenclature used in the text. Only the head groups are shown for BChl-b, BPh-b, and qui nones; the carotenoid was
omitted.
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
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DEISENHOFER & MICHEL
amino acid residues, four histidines (Ll90, L230, M217, M264), and one glutamic acid (M232) (29, 70). The Mg2+ ions of the BChl-b are five coordinated with two histidine residues each from the subunits L and M forming metal-nitrogen bonds (Ll 53 ---> BA, Ll73 ---> DL, M180 ---> BB, M200 ---> DM)' DL, DM,
Further
Quick links to online content
Annll, ReI). BioplJys. Biopl1rs. Chern. 1991. 20:247-{)6 Copyright © 1991 by Ann�al Reviews Inc. All rights resen'ed
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
HIGH-RESOLUTION STRUCTURES OF PHOTOSYNTHETIC REACTION CENTERS Johann Deisenhofer Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Hartmut Michel Max-Planck-Institut fiir Biophysik, Abteilung Molekulare Membranbiochemie, Frankfurt/Main, FRG KEY WORDS:
membrane protein structurc, RllOdospeudomonas viridis, Rhodo bacler sphaeroides, X-ray crystallography, photosynthetic purple bacteria
CONTENTS PERSPECTIVES AND OVERVIEW ..
248
CRYSTALLIZATION AND X-RAY STRUCTURE ANALYSIS OF Res ..
249
The RC from Rhodopseudomonas viridis .. The RC from Rhodobacter sphaeroides .....
250 257 258
IMPLlCATIONS..
PUlhway.i and Mechanism of Electron Transfer RC Mutants. .... ........... ....... .... Phnlosystem 1I RC: Similarity to RCs of Purple Bacteriu .... Membrane Protein Structure ........ . ..... .......
..
.
..
...
261
PROSPECTS ..
Crystallography .
.
258 259 260 260 261 263
.
Eleclrot1 Microscopy..
247 0883-9182/91/061 0--{)247$02.00
248
DEISENHOFER & MICHEL
PERSPECTIVES AND OVERVIEW Photosynthetic reaction centers (RCs) 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. Absorption of the energy of a photon in the RC is followed by rapid,
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
efficient electron transfer from a primary donor along a chain of acceptors leading across the photosynthetic membrane. [n RCs from photosynthetic purple bacteria, or from photosystem II of green plants and cyanobacteria , the end of this chain is a quinone that, after double reduction and pro tonation converts it into a quinol, dissociates from the RC. The end of the electron-transfer chain in photosystem I is an iron-sulphur center, and the small, water-soluble, electron-transport protein ferredoxin (47). Photosynthetic purple bacteria were the first organisms in which RCs were discovered (37); their relative simplicity made them logical targets for studying photosynthetic light reactions. Reed & Clayton (87) reported the first preparation of an RC, that from Rhodopseudomonas sphaeroides (later renamed Rhodobacter sphaeroides). In spite of rapid progress with photosystems of green plants and cyano bacteria, the RCs of photosynthetic purple bacteria are still the best known mediators of primary photosynthetic processes. In particular, only two high-resolution three-dimensional structures of RCs have been deter mined; both are from purple bacteria. RCs 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 (40). Subunits Land M have very hydrophobic amino acid sequences, in which five membrane-spanning segments can be distinguished (17, 72, 109, 110, 115); subunit H has a single membrane-spanning segment near its amino ter minus (73, 108, 115). The subunits Land M bind bacteriochlorophylls (BChl), bacteriopheophytins (BPh), quinones, a ferrous iron ion, and a carotenoid as cofactors. Species differ in the amino acid sequences of their protein subunits and in the types of cofactors bound to them. Amino acid sequence homologies between subunits from four different species range from 60 to 78% for subunit L, sequences of three H-subunits show homologies between 38 and 64% (108). BChl and BPh of type a (BChl-a, BPh-a) are found e.g. in RCs from
R. sphaeroides and Rhodobacter capsula/us; type b (BChl-b and BPh-b) are e.g. in Rhodopseudomonas viridis and Thiocapsa pfennigii (91). The quinones also can be of different types: R. sphaeroides has two ubiquinone-
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
REACTION CENTER STRUCTURES
249
10 molecules (39), whereas R. viridis has one menaquinone-9 and one ubiquinone-9 molecule (46). Various types of carotenoids are used in different species; for example in R. viridis, the major carotenoid species is 1,2-dihydroneurosporene (I. Sinning & H. Michel, unpublished data); in R. sphaeroides, it is spheroidene (21). Another major difference between RCs from different species of photo synthetic purple bacteria is the presence or absence of a four-heme c-type cytochrome as the fourth protein subunit. Such a subunit is part of the RC from e.g. R. viridis and T. pfennigii; RCs of other bacteria, e.g. R. sphaeroides, R. capsulatus, and Rhodospirillum rubrum, consist of only the three protein subunits L,M, and H. In this review, we focus on X-ray crystallographic studies on RCs from purple bacteria and briefly describe their structures using the RC from R. viridis as an example. We then discuss implications of the structures for RC function, relations to photosystem II, 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 RCs: structure and function (16, 39, 83), spectroscopy and electron transfer (12, 43), site-specific mutagenesis (23), primary photochemistry (16, 55), and membrane protein structure (69, 89). Useful collections of artieles on these topics can also be found in conference proceedings ( 1 5, 74). CRYSTALLIZATION AND X-RAY STRUCTURE ANALYSIS OF RCs
To date, high-resolution structural information on RCs has been obtained only by using X-ray crystallography. By "high resolution," we mean res olution sufficient to construct a reasonably accurate atomic model of a molecule; the resolution needed for a reliable model dcpcnds on thc quality and completeness of the diffraction data and on the accuracy of the phase information available; it usually lies between 3.5 and 3.0 A. A prerequisite 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 generally considered impossible. Then three-dimensional crystals were obtained for bacteriorhodopsin (7 1 ) and matrix porin from Escherichia coli (45) using the detergent octylglucoside to form water soluble protein-detergent complexes that could be treated like soluble proteins in vapor diffusion experiments in which ammonium sulphate or
250
DEISENHOFER & MICHEL
polyethylene glycol served as precipitants. These experiments were soon followed by the crystallization (65) and X-ray structure analysis (28) of the RC from R. viridis. The RC from Rhodopseudomonas viridis
The RC from the purple bacterium R. viridis is a complex of 4 protein subunits and 14 cofactors. The protein subunits are, in order of decreasing size, cytochrome [336 amino acids (a.a.)], subunit M (323 a.a.), subunit L (273 a.a.) and subunit H (258 a.a.); the complete amino acid sequences of these subunits were derived from the sequences of the genes coding for them (72, 73, 106). The cofactors are four heme groups, covalently linked to the cytochrome subunit, four BChl-b, two BPh-b, a menaquinone-9 and a ubiquinone-9, a ferrous iron ion, and the carotenoid dihydroneurosporene (46, 97).
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
COMPOSITION
Well-ordered three-dimensional crystals of the RC from R. were obtained from RCs solubilized with the detergent N,N-di methyl-dodecylamine-N-oxide (LDAO); the small amphiphile heptane1,2,3-triol was an essential additive, and ammonium sulphate was the precipitant in a vapor-diffusion experiment (65, 66). The crystals have the symmetry of space group P432]2. The unit cell constants are a b 223.5 A, c = 113.6 A, and 8 RC molecules per unit cell (28, 65). The crystals diffract X-rays to at least 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 (28). CRYSTALS
viridis
=
=
The structure of the RC crystals was initially determined at 3.0-A resolution using the method of multiple isomorphous replacement with heavy atom compounds (28). Small mercury compounds and uranyl nitrate were especially useful for that purpose. To preserve the high crystalline order during the soaking experiments, an additional purification step had to be introduced, and a special "soak buffer" had to be developed (28, 31). Phase information from five heavy atom derivatives enabled the calculation of an electron density map, which the method of solvent flattening (104) further improved. On the basis of this electron density map, an atomic model of the RC was built for the cofactors (28) and the protein subunits (29). The combination of sequence information and crystallographic data allowed the description of protein cofactor inter actions (70). Crystallographic refinement at 2.3-A resolution (1. Deisen hofer, O. Epp, I. Sinning, & H. Michel, in preparation) led to an atomic model consisting of 10,288 atoms, including e.g. 201 bound water molecules, 7 bound anions, and 1 firmly bound LDAO molecule (30-32). The upper limit for the average error in atomic coordinates (62) was X-RAY STRUCTURE ANALYSIS
REACTION CENTER STRUCTURES
251
estimated as 0.266 A (31, 32). The X-ray data did not allow distinction among carbon, nitrogen, and oxygen atoms. Orientations of side chains of amino acids such as asparagine, glutamine, histidine, and threonine, or of acetyl groups of BChl and BPh, were chosen to interact optimally with the environment. In a low-resolution neutron diffraction study, the distribution of the detergent in the crystal could be determined (90). Figure I shows an overall view of the model of the RC from 130 A; the maximum width perpendicular to that direction is 70 A. The closely associated subunits L and M, together with the bound Behl, BPh, quinones, nonheme iron, and carotenoid, form the central part of the RC. The most 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 (90), by properties of the model (31), and by functional considerations. The
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
RC MODEL
R. viridis. In its longest dimension, the complex measures
�
'"
Figure 1
(Stereo pair) Overall view of the RC from R. viridis. The polypeptide chains of
the protein subunits are represented by ribbons; co factors are drawn as wire models. The membrane plane is approximately perpendicular to the plane of projection. The approximate positions of the periplasmic (upper line) and the cytoplasmic (lower line) membrane surfaces are indicated.
Annu. Rev. Biophys. Biophys. Chem. 1991.20:247-266. Downloaded from www.annualreviews.org Access provided by KTH Royal Institute of Technology (Sweden) on 11/14/15. For personal use only.
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polypeptide backbones of subunits L and M and the attached cofactors display a high degree of local two-fold symmetry; the local two-fold sym metry axis is oriented perpendicular to the membrane plane (13). 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 at the periplasmic side, and the globular domain of the H-subunit is at the cytoplasmic side of the membrane. The H-subunit contributes a single membrane-spanning helix. Neither the cytochrome nor the H-subunit obey the local symmetry found in the central part of the RC; the cytochrome has internal local symmetry of its own. Figure 2 shows schematic drawings of the polypeptide-chain folding of the reaction center subunits L and M; the structural similarity between these subunits is obvious. Structurally similar segments in the L and M subunits include the transmembrane helices (labeled A, B, C, D, E) and a large fraction of the connections. In total, 216 IJ. carbons from the M subunit can be superimposed onto corresponding IX carbons of the L subunit by a rotation of almost exactly 1800 to a root mean square (rms) deviation of 1.22 A. In addition to the transmembrane helices, which have lengths between 21 and 28 residues, the connecting segments contain shorter helices. The M subunit is 50 residues longer than the L subunit. Both sequence alignments (72) and structural alignments (29, 31) show that the sequence insertions in M reside on either side of the trans membrane helices (20 at the NH2 terminus, 7 in the connection between transmembrane helices MA and MB, 7 in the connection between MD and ME, and 16 at the COOH terminus). Through these insertions, the M subunit dominates the contacts of the L-M complex to the peripheral subunits. The insertion between transmembrane helices MD and ME, containing another small helix, is of importance 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 structural regions with different characteristics. The amino terminal segment, begin ning with formylmethionine (73), contains the only transmembrane helix of subunit H (residues H l 2 to H35). Near the end of the transmembrane helix, the sequence shows seven consecutive charged residues (H33 to H39). Residues H47 to HS3 are disordered in the crystal so that no significant electron density can be found for them. Following the dis ordered region, the H chain forms an extended structure along the surface of the L -M complex, apparently deriving structural stability from that contact. The surface region contains a short helix and two two-stranded antiparallel f3-sheets. The third structural segment of the H-subunit, start ing at about H l OS, forms a globular domain. This domain contains an
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REACTION CENTER STRUCTURES
MBO
Figure 2
(Stereo
253
MBO
pairs) L-subunit (top) and M-subunit (bottom) of the RC from R. viridis
in ribbon representation. The transmembrane helices in each subunit are labeled A, B, C, D, and E.
extended system of parallel and antiparallel /I-sheets (between residues H134 and H203), and an a-helix (residues H232 to H248). The cytochrome is the largest subunit in the reaction center complex
(106).
Its structure, shown in Figure 4, consists of an NHz-terminal
segment, two pairs of heme-binding segments, and a segment connecting the two pairs. Each heme binding segment consists of a helix with an
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Figure 3
(Stereo pair) The H-subunit of the RC from R. viridis in ribbon representation.
Residues H47 to H53 are disordered in the crystal and not shown.
average length of 17 residues followed by a turn and the Cys-X-y-Cys His sequence typical for c-type cytochromes. The hemes are connected to the cysteine residues via thioether linkages. This arrangement makes the heme planes parallel to the helix axes. The sixth ligand to the heme iron is a methionine residue within the helix in three of the four cases. Histidine C124, located in a different part of the structure, serves as a sixth ligand for the iron of heme 4. The two pairs of heme binding segments, containing hemes I and 2 and 3 and 4, respectively, are related by a local two-fold symmetry. Sixty-five residues of each pair obey this local symmetry with an rms deviation between corresponding IX carbon atoms of 0.93 A. The local symmetry of the cytochrome is not related to the central local sym metry. The last four residues, C333 to C336, are disordered. Also dis ordered is the lipid moiety bound to the NH2-terminal cysteine residue (107). Figure 5 shows arrangements and nomenclature of the cofactors associ ated with the protein subunits L and M, excluding phytyl side chains or isoprenoid side chains for clarity. A closely associated pair of BChl-bs, the special pair, resides at the origin of two branches of cofactors, each of which consists of another BChl-b (the accessory BChl-b), a BPh-b, and a quinone. The nonheme iron sits between the quinones and is bound to five
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REACTION CENTER STRUCTURES
Figure 4
255
(Stereo pair) The cytochrome subunit of the RC from R. viridis. The polypeptide
chain is drawn as a ribbon; the hemes and the heme-binding cysteine residues are drawn as wire models. The view is approximately along the local two-fold symmetry axis of the cytochrome. The hemes are numbered 1,2,4,3 (top to bottom; numbers not shown).
Figure 5
(Stereo pair) Model of the co factors associated with subunits L and M in the RC
from R. viridis. The molecules are labeled according to the nomenclature used in the text. Only the head groups are shown for BChl-b, BPh-b, and qui nones; the carotenoid was
omitted.
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amino acid residues, four histidines (Ll90, L230, M217, M264), and one glutamic acid (M232) (29, 70). The Mg2+ ions of the BChl-b are five coordinated with two histidine residues each from the subunits L and M forming metal-nitrogen bonds (Ll 53 ---> BA, Ll73 ---> DL, M180 ---> BB, M200 ---> DM)' DL, DM,
