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The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis — An overview and recent advances
Biochimica et BiophysicaActa, 1018 (1990) 115-118 Elsevier
115
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The photosynthetic reaction centre from the purple bacterium R h o d o p s e u d o m o n a s c i r i d i s - an overview and recent advances Hartmut Michel, Irmgard Sinning, Juergen Koepke, Gunther Ewald and Giinter Fritzsch (Received 1 May 1990)
Key words: Photosynthesis;Reaction center; Membraneprotein; (Rps. oiridis) Introduction
In photosynthesis the primary charge separation and the subsequent electron transfer across the photosynthetic membrane occur in the so-called photosynthetic reaction centres. The photosynthetic reaction centres consist of integral membrane proteins which contain the binding sites for the photosynthetic pigments. These are involved in the initial light absorption, or the energy transfer from the surrounding light-harvesting pigments, leading to the primary charge separation, and the subsequent electron transfer. The best known reaction centres are those from the purple photosynthetic bacteria (for review see Refs. 1 and 2). They are also the only membrane proteins or membrane protein complexes which have yielded crystals that allowed the performance of a high resolution X-ray structure determination. Most of the reaction centres from purple bacteria contain three protein subunits which are called H (heavy), M (medium) and L (light) subunits according to their apparent molecular weights as determined by sodium dodecylsulphate polyacrylamide gel electrophoresis. The reaction centres from Rhodopseudomonas (Rps.) viridis contain as a fourth subunit a tightly bound cytochrome molecule with four haem groups. The cytochrome subunit is involved in the re-reduction of the photo-oxidized primary electron donor. The photosynthetic pigments and cofactors in the Rps. viridis reaction centre are four bacteriochlorophyll b molecules, two bacteriopheophytin b molecules, one menaquinone as primary quinone ('QA'), one non-haem-iron and one ubiquinone as secondary quinone ('QB'). The successful crystallization of the reaction centre [3] opened the way to the determination of its three-dimensional structure [4-6]. In this paper we will shortly summarize the
Correspondence: H. Michel, Max-Planck-Institut f'gr Biophysik, Abteilung Molekulare Membranbiologie, Heinrich-Hoffmann-Str.7, 6000 Frankfurt/M 71, F.R.G.
pigment arrangement and protein structure. A more detailed view is presented in Ref. 7. Recent results concerning the correlation of the haem groups with respect to position, redox potential and spectroscopic properties, and the characterization of herbicide-resistant mutants are presented. Structural overview
Pigment arrangement The two major surprises of the structure analysis were the discovery of the quasisymmetric structure of the core of the reaction centre, and the linear arrangement of the four haem groups. The arrangement of the pigments and cofactors is shown in Fig. 1 (taken from Ref. 4). The four haem groups are seen at the top in Fig. 1. The function of these haems is to re-reduce the photooxidized primary electron donor which is a 'dimer' of two non-covalently linked bacteriochlorophyll molecules ('special pair'). The ring systems of the two bacteriochlorophylls constituting the dimer are nearly parallel and in projection they overlap with their pyrrole rings I. The plane-to-plane distance of these two bacteriochlorophylls is about 3.1 ,A. The ring systems of all the chlorine pigments are related by an approximate two-fold rotation axis. This diad (vertically in Fig. 1) runs through the special pair on the periplasmic side of the membrane and through the non-haem iron on the cytoplasmic side of the membrane. Since the two 'accessory' bacteriochlorophylls and the two bacteriopheophytins are also related by the local diad, two structurally equivalent branches are formed which could be used for electron transfer across the membrane. However, only one of these is used, as indicated by the fact that the two bacteriopheophytins are spectroscopically inequivalent by absorbing light of different wavelengths, and only the one absorbing light at the longer wavelengths is an intermediate electron acceptor. Comparison of absorbance spectra of crystals taken with polarized light shows that the bacteriopheophytin absorbing at the longer wavelength is closer to Q^. In
0005-2728/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
116
0
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•
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•
•
•
•
g
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•
•
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MQ
Fig. 1. (Stereo pair.) Arrangement of the pigments in the photosynthetic reaction centre from Rps. viridis, showing from top to bottom four haem groups (HE), fou~" bactedoehlorophylls (BC), one non-haem iron (Fe), and one menaquinone (MQ). The approximate two-fold symmetry axis relating the photosynthetic pigments runs vertically in the plane of the picture. The approxirclate position of the periplasmic and cytopL'.smic face of the photosynthetic membrane is indicated by dotted lines. Taken from Ref. 4.
addition, most of the Qs is lost during the isolation and crystallization of the reaction centre, QB could be local-ized in the electron density m~.p only during the refinement of the structure at 2,3 A resolution (Deisenhofer, J., Epp, O., Sinning, I. and Michel, H., unpublished data). These results establish cl,afly that only the pigment branch shown on the right-hand side in Fig. 1 is used for the light-driven electron transfer across the membrane. It is more closely associated with the L-subunit. The binding site of Qs is symmetrically related by the local diad to the binding site of QA" This means that the electron has to be transferred from QA to Qs parallel to the surface of the membrane. There is no evidence for the participation of the non-haem iron in this electron transfer, since in Rhodobacter (Rb.) sphaeroides it can be removed without changing the kinetics of this electron transfer [8]. The role of the accessory bacteriochlorophyll is under debate. Until last year the accepted view was that the first electron acceptor is the bacteriopheophytin on the L-side (see Refs. 9 and 10). The situation has changed now, since Zinth and co-workers [11] provided evidence that the accessory chlorophyll is the first electron acceptor and that the electron is then transferred to the bacteriopheophytin. This second electron transfer is 4-times faster than the first one [11].
Protein structure and protein-pigment interactions A drawing of the polypeptide chains together with the chromophore model is shown in Fig. 2 (taken from Ref. 5). The L- and M subunits bind the pigments and form the central part of the reaction centre. The L- and M-subunits are of similar structure, each possessing five long membrane-spanning helices which are related by the same two-fold axis as the pigments. When compared to the M-subunit the L-subunit possesses insertions at the carboxy- and amino-termini and in the connection of the first and second transmembrane helices as well as in the connection of the fourth and fifth transmembrane helices. On both sides of the helical regions of the Land M-subunits the polypeptide segments connecting the transmembrane helices and the terminal segments form flat surfaces. The cytochrome subunit is bound to the surface close to the special paint Q)a the periplasmic side. The H-subunit possesses one membrane-spanning helix close to its amino-terminus. Its large carboxyterminal domain is bound to the flat surface of the L-, M-complex on the cytoplasmic side. The photosynthetic pigments are bound into primarily hydrophobic pockets of the L- and M-subunits [6]. All the bacteriochlorophylls possess histidine residues as ligands to their magnesium atoms, which are all five coordinated. The protein must specifically interact with
117 the pigments in order to suppress one of the two possible electron transport pathways. Most likely the decision is already made in the vicinity of the special pair. The distribution of polar side chains and the hydrogen bonding pattern is different between both pigment branches already around the special pair, but also around the bacteriopheophytins. The primary quinone is bound into a rather tight hydrophobic pocket. Tryptophan M250 forms the major part of its binding site. The binding site of the secondary quinone is considerably larger and more polar in agreement with the role of the secondary quinone as a mobile electron donor which has to leave the reaction centre after reduction by two electrons and protonation. Glutamic acid L212 seems to be involved in this protonation. The binding site of QB is also the site of action of some herbicides (see below) which act by displacing the secondary quinone, thereby stopping the light-induced electron flow. A s s i g n m e n t o f the h a e m groups
The a bands of the four haem groups possess different absorption maxima and redox potentials. Only the two haem groups having high midpoint redox potentials can be reduced by the water-soluble cytochrome c 2. Based on optical spectroscopy or EPR measurements proposals for the arrangement of the haem groups and
TABLE I Correlation of position, optical properties and redox potentials of the haem groups in the photosynthetic reaction centre from Rps. viridis
Haem 1 Haem 2 Haem 4 Haem 3
Absorption maximum (rim) 552.5 556 552 558.5
Special pair
960
Midpoint potential (mV) - 60 + 300 + 10 + 370
the assignments of optical properties and redox potentials have been made [12-14]. A firm proof for the assignments could be obtained by studying in crystals the haem group orientation with polarized light under variation of the redox potentials [15]. The results are given in Table I. Haem 3 is closest to the primary electron donor and has the highest redox potential. Haem 4 is located next to haem 3 and has a low re(lox potential, followed by haem 2 which has a high redox potential. At the top haem 1 is located which has the lowest redox potential. The low redox potential haems cannot be involved in the light-driven cyclic electron flow. Their function is unknown and the meaning of the
Fig. 2. (Stereo pair.) Ribbon drawing of the polypeptidechains of the reaction centre from Rps. viridis, together with the chromophoremodel (thin lines), showing the cytochrome subunit (top), and the subunits L (middle left), M (middle right), and H (bottom, with the N-terminal helix extending from the cytochrome).Taken from Ref. 5.
118 a l t e r n a t e o r d e r of high a n d low r e d o x p o t e n t i a l s has to b e clarified. Characterization o f herbicide-resistent mutants D u e to their e v o l u t i o n a r y relation a n d f u n c t i o n a l equivalence the r e a c t i o n centres of p h o t o s y s t e m II a n d f r o m p u r p l e b a c t e r i a are b o t h the target of herbicides of the s-triazine-type. E x a m p l e s are atrazine (2-chloro-4(ethylarnino)-6-(isopropylamino)-s-triazine) and terbut r y n (2-(methylthio)-4-(ethylamino)-6-( tert-butylamino)-5-triazine). Several m u t a n t s o f Rps. viridis resistant to these h e r b i c i d e s have b e e n selected [16,17]. A l l m u t a n t s c a r r y m u t a t i o n s in the vicinity of the Q a - b i n d ing site, b u t in one of the m u t a n t s the m u t a t e d a m i n o a c i d ( L 2 2 2 T y r ~ Phe) is n o t in c o n t a c t with the herbicide t e r b u t r y n whose p o s i t i o n in the r e a c t i o n centre has b e e n d e t e r m i n e d b y X - r a y c r y s t a l l o g r a p h y [6]. D e s p i t e the very similar c h e m i c a l structure, m o s t of the m u t a tions l e a d i n g to resistance are different for b o t h herbicides. T h e structures of m u t a n t s T1, T4 a n d T5 have b e e n s t u d i e d b y X - r a y c r y s t a l l o g r a p h y using difference F o u r i e r techniques, a n d have b e e n refined. M u t a n t T1 is a d o u b l e m u t a n t (L217 A r g ~ His, L223 Ser ~ Ala). I n the w i l d - t y p e the side-chain o f L223 Ser accepts a h y d r o g e n b o n d f r o m the a m i n o e t h y l side-chain of t e r b u t r y n . Loss of this h y d r o g e n b o n d is sufficient to e x p l a i n the h e r b i c i d e resistance. T h e X - r a y structure analysis shows t h a t the side-chain of L213 A s n which is also h y d r o g e n - b o n d e d to the side-chain of L223 Ser m o v e s into a new p o s i t i o n [20]. This is p o s s i b l e since the r e p l a c e m e n t of L217 A r g b y the smaller His p r o v i d e s a d d i t i o n a l space for the side-chain of L213 Asn. I n m u t a n t T1 the changes in the electron d e n s i t y are small a n d well defined. A n o p p o s i t e result has b e e n o b t a i n e d for m u t a n t T4 (L222 T y r ~ Phe). In the w i l d - t y p e this t y r o s i n e forms a h y d r o g e n b o n d with the b a c k b o n e o f M41. A s a result of the loss of this h y d r o g e n b o n d , the side-chain o f L222 changes its p o s i t i o n a n d moves into the Q a - b i n d i n g site which w i d e n s to a c c o m m o d a t e the new p h e n y l a l a n i n e side-chain. A s a very d r a m a t i c c h a n g e a m i n o acids M 2 5 - M 5 5 also m o v e to a new position. A s an a d d i t i o n a l result of the a l t e r n a t i o n of the Q B - b i n d i n g site this m u t a n t n o w b e c o m e s sensitive t o w a r d s the plant herbicide DCMU (3-(3,4-dichlorophenyl)-l,l-dimethylurea), also k n o w n as d i u r o n [18]. E P R s p e c t r a of Q ~ in this m u t a n t also show a higher similarity to P h o t o s y s t e m II w h e n c o m p a r e d to w i l d - t y p e [17]. T h e r e f o r e this m u t a n t seems to p r o v i d e an excellent m o d e l system for P h o t o s y s t e m II. A m o n g the m u t a n t s resistant t o w a r d s atrazine, the m u t a t i o n L212 G l u ~ Lys is the m o s t interesting one. T h e structure suggests the i n v o l v e m e n t of this residue in the p r o t o n a t i o n of QB [6]. E x p e r i m e n t a l evidence for this p r o p o s a l is n o w available for Rb. sphaeroides [19]. It will b e of great interest to c h a r a c t e r i z e this m u t a n t in detail.
TABLE II Herbicide-resistant mutants from Rps. viridis selected by their resistance towards terbutryn (T) or atrazine (.4) and the location of their mutation
Terbutryn-resistant: T1 = T2 T3 T4 T5 = T6 = T7
L217 Arg ~ L216 Phe ~ L217 Tyr ~ L216 Phe ~
Atrazine-resistant: A2 A3 = T5, T6, T7 A4 = A5
L212 Glu ~ Lys L216 Phe --*SCr L217 Arg ~ His, L220 Val ---,Leu
His, L223 Ser ~ Ala Ser, M263 Val ~ Phe Phe Ser
References 1 Feher, G., Allen, J.P., Okamura, M.Y. and Rees, D.C. (1989) Nature 339, 111-116. 2 Hoff, A.I. (1982) in Molecular Biology, Biochemistry and Biophysics 35 (Fong, F.U., ed.), pp. 81-151, Springer, Berlin. 3 Michel, H. (1982) J. Mol. Biol. 158, 567-572. 4 Deisenhofer, J., Epp. O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 358-389. 5 Deisenhofer, J., Epp, O., Mild, K., Huber, R. and Michel, H. (1985) Nature 318, 618-624. 6 Michel, H., Epp, G. and Deisenhofer, J. (1986) EMBO J. 5, 2445-2461. 7 Deisenhofer, J. and Michel, H. (1989) EMBO J. 8, 2149-2169 (Nobel Lecture). 8 Debus, R.J., Okamura, M.Y. and Feher, G. (1985) Biophys. J. 47, 3a. 9 Breton, J., Martin, J.L., Fleming, G.R. and Lambry, J.-C. (1988) Biochemistry 27, 8276-8284. 10 Fleming, G.R., Martin, J.L and Breton, J. (1988) Nature 333, 190-192. 11 Holzapfel, W., Finkele, U., Kaiser, W., Oesterholt, D., Scheer, H., Stilz, H.U. and Zinth, W. (1989) Chem. Phys. Lett. 160, 1-7. 12 Dracheva, S.M., Drachev, L.A., Konstantinov, A.A., Semenov, A.Yu., Skulachev, V.P., Arutjunjan, A.M., Shuvalov, V.A. and Zaberezhnaya, S.M. (1988) Eur. J. Biochem. 171,253-264. 13 Nitschke, W. and Rutherford, A.W. (1989) Biochemistry 28, 31613168. 14 Vermeglio, A., Richaud, P. and Breton, J. (1989) FEBS Lett. 243, 259-263. 15 Fritzsch, G., Buchanan, S. and Michel, H. (1989) Biochim. Biophys. Acta 977, 157-162. 16 Sinning, I., Michel, H., Mathis, P. and Rutherford, A.W. (1989) Biochemistry 28, 5544-5553. 17 Ewald, G., Wiessner, C. and Michel, H. (1990) Z. Naturforsch. 45C, 459-462. 18 Sinning, I., Michel, H., Mathis, P. and Rutherford, A.W. (1989) FEBS Lett. 256, 192-194. 19 Paddock, M.L., Rongey, S.H., Feher, (3. and Okamura, M.Y. (1989) Proc. Natl. Acad. Sci. USA 86, 6602-6606. 20 Sinning, I., Koepke, J., Schiller, G. and Michel, H. (1990) Z. Naturforsch. 45C, 455-458.
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The photosynthetic reaction centre from the purple bacterium R h o d o p s e u d o m o n a s c i r i d i s - an overview and recent advances Hartmut Michel, Irmgard Sinning, Juergen Koepke, Gunther Ewald and Giinter Fritzsch (Received 1 May 1990)
Key words: Photosynthesis;Reaction center; Membraneprotein; (Rps. oiridis) Introduction
In photosynthesis the primary charge separation and the subsequent electron transfer across the photosynthetic membrane occur in the so-called photosynthetic reaction centres. The photosynthetic reaction centres consist of integral membrane proteins which contain the binding sites for the photosynthetic pigments. These are involved in the initial light absorption, or the energy transfer from the surrounding light-harvesting pigments, leading to the primary charge separation, and the subsequent electron transfer. The best known reaction centres are those from the purple photosynthetic bacteria (for review see Refs. 1 and 2). They are also the only membrane proteins or membrane protein complexes which have yielded crystals that allowed the performance of a high resolution X-ray structure determination. Most of the reaction centres from purple bacteria contain three protein subunits which are called H (heavy), M (medium) and L (light) subunits according to their apparent molecular weights as determined by sodium dodecylsulphate polyacrylamide gel electrophoresis. The reaction centres from Rhodopseudomonas (Rps.) viridis contain as a fourth subunit a tightly bound cytochrome molecule with four haem groups. The cytochrome subunit is involved in the re-reduction of the photo-oxidized primary electron donor. The photosynthetic pigments and cofactors in the Rps. viridis reaction centre are four bacteriochlorophyll b molecules, two bacteriopheophytin b molecules, one menaquinone as primary quinone ('QA'), one non-haem-iron and one ubiquinone as secondary quinone ('QB'). The successful crystallization of the reaction centre [3] opened the way to the determination of its three-dimensional structure [4-6]. In this paper we will shortly summarize the
Correspondence: H. Michel, Max-Planck-Institut f'gr Biophysik, Abteilung Molekulare Membranbiologie, Heinrich-Hoffmann-Str.7, 6000 Frankfurt/M 71, F.R.G.
pigment arrangement and protein structure. A more detailed view is presented in Ref. 7. Recent results concerning the correlation of the haem groups with respect to position, redox potential and spectroscopic properties, and the characterization of herbicide-resistant mutants are presented. Structural overview
Pigment arrangement The two major surprises of the structure analysis were the discovery of the quasisymmetric structure of the core of the reaction centre, and the linear arrangement of the four haem groups. The arrangement of the pigments and cofactors is shown in Fig. 1 (taken from Ref. 4). The four haem groups are seen at the top in Fig. 1. The function of these haems is to re-reduce the photooxidized primary electron donor which is a 'dimer' of two non-covalently linked bacteriochlorophyll molecules ('special pair'). The ring systems of the two bacteriochlorophylls constituting the dimer are nearly parallel and in projection they overlap with their pyrrole rings I. The plane-to-plane distance of these two bacteriochlorophylls is about 3.1 ,A. The ring systems of all the chlorine pigments are related by an approximate two-fold rotation axis. This diad (vertically in Fig. 1) runs through the special pair on the periplasmic side of the membrane and through the non-haem iron on the cytoplasmic side of the membrane. Since the two 'accessory' bacteriochlorophylls and the two bacteriopheophytins are also related by the local diad, two structurally equivalent branches are formed which could be used for electron transfer across the membrane. However, only one of these is used, as indicated by the fact that the two bacteriopheophytins are spectroscopically inequivalent by absorbing light of different wavelengths, and only the one absorbing light at the longer wavelengths is an intermediate electron acceptor. Comparison of absorbance spectra of crystals taken with polarized light shows that the bacteriopheophytin absorbing at the longer wavelength is closer to Q^. In
0005-2728/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
116
0
•
•
•
•
•
•
•
•
g
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•
•
•
•
MQ
Fig. 1. (Stereo pair.) Arrangement of the pigments in the photosynthetic reaction centre from Rps. viridis, showing from top to bottom four haem groups (HE), fou~" bactedoehlorophylls (BC), one non-haem iron (Fe), and one menaquinone (MQ). The approximate two-fold symmetry axis relating the photosynthetic pigments runs vertically in the plane of the picture. The approxirclate position of the periplasmic and cytopL'.smic face of the photosynthetic membrane is indicated by dotted lines. Taken from Ref. 4.
addition, most of the Qs is lost during the isolation and crystallization of the reaction centre, QB could be local-ized in the electron density m~.p only during the refinement of the structure at 2,3 A resolution (Deisenhofer, J., Epp, O., Sinning, I. and Michel, H., unpublished data). These results establish cl,afly that only the pigment branch shown on the right-hand side in Fig. 1 is used for the light-driven electron transfer across the membrane. It is more closely associated with the L-subunit. The binding site of Qs is symmetrically related by the local diad to the binding site of QA" This means that the electron has to be transferred from QA to Qs parallel to the surface of the membrane. There is no evidence for the participation of the non-haem iron in this electron transfer, since in Rhodobacter (Rb.) sphaeroides it can be removed without changing the kinetics of this electron transfer [8]. The role of the accessory bacteriochlorophyll is under debate. Until last year the accepted view was that the first electron acceptor is the bacteriopheophytin on the L-side (see Refs. 9 and 10). The situation has changed now, since Zinth and co-workers [11] provided evidence that the accessory chlorophyll is the first electron acceptor and that the electron is then transferred to the bacteriopheophytin. This second electron transfer is 4-times faster than the first one [11].
Protein structure and protein-pigment interactions A drawing of the polypeptide chains together with the chromophore model is shown in Fig. 2 (taken from Ref. 5). The L- and M subunits bind the pigments and form the central part of the reaction centre. The L- and M-subunits are of similar structure, each possessing five long membrane-spanning helices which are related by the same two-fold axis as the pigments. When compared to the M-subunit the L-subunit possesses insertions at the carboxy- and amino-termini and in the connection of the first and second transmembrane helices as well as in the connection of the fourth and fifth transmembrane helices. On both sides of the helical regions of the Land M-subunits the polypeptide segments connecting the transmembrane helices and the terminal segments form flat surfaces. The cytochrome subunit is bound to the surface close to the special paint Q)a the periplasmic side. The H-subunit possesses one membrane-spanning helix close to its amino-terminus. Its large carboxyterminal domain is bound to the flat surface of the L-, M-complex on the cytoplasmic side. The photosynthetic pigments are bound into primarily hydrophobic pockets of the L- and M-subunits [6]. All the bacteriochlorophylls possess histidine residues as ligands to their magnesium atoms, which are all five coordinated. The protein must specifically interact with
117 the pigments in order to suppress one of the two possible electron transport pathways. Most likely the decision is already made in the vicinity of the special pair. The distribution of polar side chains and the hydrogen bonding pattern is different between both pigment branches already around the special pair, but also around the bacteriopheophytins. The primary quinone is bound into a rather tight hydrophobic pocket. Tryptophan M250 forms the major part of its binding site. The binding site of the secondary quinone is considerably larger and more polar in agreement with the role of the secondary quinone as a mobile electron donor which has to leave the reaction centre after reduction by two electrons and protonation. Glutamic acid L212 seems to be involved in this protonation. The binding site of QB is also the site of action of some herbicides (see below) which act by displacing the secondary quinone, thereby stopping the light-induced electron flow. A s s i g n m e n t o f the h a e m groups
The a bands of the four haem groups possess different absorption maxima and redox potentials. Only the two haem groups having high midpoint redox potentials can be reduced by the water-soluble cytochrome c 2. Based on optical spectroscopy or EPR measurements proposals for the arrangement of the haem groups and
TABLE I Correlation of position, optical properties and redox potentials of the haem groups in the photosynthetic reaction centre from Rps. viridis
Haem 1 Haem 2 Haem 4 Haem 3
Absorption maximum (rim) 552.5 556 552 558.5
Special pair
960
Midpoint potential (mV) - 60 + 300 + 10 + 370
the assignments of optical properties and redox potentials have been made [12-14]. A firm proof for the assignments could be obtained by studying in crystals the haem group orientation with polarized light under variation of the redox potentials [15]. The results are given in Table I. Haem 3 is closest to the primary electron donor and has the highest redox potential. Haem 4 is located next to haem 3 and has a low re(lox potential, followed by haem 2 which has a high redox potential. At the top haem 1 is located which has the lowest redox potential. The low redox potential haems cannot be involved in the light-driven cyclic electron flow. Their function is unknown and the meaning of the
Fig. 2. (Stereo pair.) Ribbon drawing of the polypeptidechains of the reaction centre from Rps. viridis, together with the chromophoremodel (thin lines), showing the cytochrome subunit (top), and the subunits L (middle left), M (middle right), and H (bottom, with the N-terminal helix extending from the cytochrome).Taken from Ref. 5.
118 a l t e r n a t e o r d e r of high a n d low r e d o x p o t e n t i a l s has to b e clarified. Characterization o f herbicide-resistent mutants D u e to their e v o l u t i o n a r y relation a n d f u n c t i o n a l equivalence the r e a c t i o n centres of p h o t o s y s t e m II a n d f r o m p u r p l e b a c t e r i a are b o t h the target of herbicides of the s-triazine-type. E x a m p l e s are atrazine (2-chloro-4(ethylarnino)-6-(isopropylamino)-s-triazine) and terbut r y n (2-(methylthio)-4-(ethylamino)-6-( tert-butylamino)-5-triazine). Several m u t a n t s o f Rps. viridis resistant to these h e r b i c i d e s have b e e n selected [16,17]. A l l m u t a n t s c a r r y m u t a t i o n s in the vicinity of the Q a - b i n d ing site, b u t in one of the m u t a n t s the m u t a t e d a m i n o a c i d ( L 2 2 2 T y r ~ Phe) is n o t in c o n t a c t with the herbicide t e r b u t r y n whose p o s i t i o n in the r e a c t i o n centre has b e e n d e t e r m i n e d b y X - r a y c r y s t a l l o g r a p h y [6]. D e s p i t e the very similar c h e m i c a l structure, m o s t of the m u t a tions l e a d i n g to resistance are different for b o t h herbicides. T h e structures of m u t a n t s T1, T4 a n d T5 have b e e n s t u d i e d b y X - r a y c r y s t a l l o g r a p h y using difference F o u r i e r techniques, a n d have b e e n refined. M u t a n t T1 is a d o u b l e m u t a n t (L217 A r g ~ His, L223 Ser ~ Ala). I n the w i l d - t y p e the side-chain o f L223 Ser accepts a h y d r o g e n b o n d f r o m the a m i n o e t h y l side-chain of t e r b u t r y n . Loss of this h y d r o g e n b o n d is sufficient to e x p l a i n the h e r b i c i d e resistance. T h e X - r a y structure analysis shows t h a t the side-chain of L213 A s n which is also h y d r o g e n - b o n d e d to the side-chain of L223 Ser m o v e s into a new p o s i t i o n [20]. This is p o s s i b l e since the r e p l a c e m e n t of L217 A r g b y the smaller His p r o v i d e s a d d i t i o n a l space for the side-chain of L213 Asn. I n m u t a n t T1 the changes in the electron d e n s i t y are small a n d well defined. A n o p p o s i t e result has b e e n o b t a i n e d for m u t a n t T4 (L222 T y r ~ Phe). In the w i l d - t y p e this t y r o s i n e forms a h y d r o g e n b o n d with the b a c k b o n e o f M41. A s a result of the loss of this h y d r o g e n b o n d , the side-chain o f L222 changes its p o s i t i o n a n d moves into the Q a - b i n d i n g site which w i d e n s to a c c o m m o d a t e the new p h e n y l a l a n i n e side-chain. A s a very d r a m a t i c c h a n g e a m i n o acids M 2 5 - M 5 5 also m o v e to a new position. A s an a d d i t i o n a l result of the a l t e r n a t i o n of the Q B - b i n d i n g site this m u t a n t n o w b e c o m e s sensitive t o w a r d s the plant herbicide DCMU (3-(3,4-dichlorophenyl)-l,l-dimethylurea), also k n o w n as d i u r o n [18]. E P R s p e c t r a of Q ~ in this m u t a n t also show a higher similarity to P h o t o s y s t e m II w h e n c o m p a r e d to w i l d - t y p e [17]. T h e r e f o r e this m u t a n t seems to p r o v i d e an excellent m o d e l system for P h o t o s y s t e m II. A m o n g the m u t a n t s resistant t o w a r d s atrazine, the m u t a t i o n L212 G l u ~ Lys is the m o s t interesting one. T h e structure suggests the i n v o l v e m e n t of this residue in the p r o t o n a t i o n of QB [6]. E x p e r i m e n t a l evidence for this p r o p o s a l is n o w available for Rb. sphaeroides [19]. It will b e of great interest to c h a r a c t e r i z e this m u t a n t in detail.
TABLE II Herbicide-resistant mutants from Rps. viridis selected by their resistance towards terbutryn (T) or atrazine (.4) and the location of their mutation
Terbutryn-resistant: T1 = T2 T3 T4 T5 = T6 = T7
L217 Arg ~ L216 Phe ~ L217 Tyr ~ L216 Phe ~
Atrazine-resistant: A2 A3 = T5, T6, T7 A4 = A5
L212 Glu ~ Lys L216 Phe --*SCr L217 Arg ~ His, L220 Val ---,Leu
His, L223 Ser ~ Ala Ser, M263 Val ~ Phe Phe Ser
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