Photosynthesis Research 48: 301-308, 1996. (~) 1996KluwerAcademic Publishers. Printedin the Netherlands. Regular paper
FTIR spectroscopy of primary donor photooxidation in Photosystem I, Heliobacillus mobUis, and Chlorobium limicola. Comparison with purple bacteria Eliane Nabedryk, Winfried Leibl & Jacques Breton Section de Bio~nerg~tique, D~partement de Biologie Cellulaire et Moldculaire, CEA/Saclay, 91191 Gif-sur-Yvette Cedex, France Received 6 December 1995; accepted in revised form 12 February 1996
Key words: bacteriochlorophyll dimer, photosynthetic reaction center, primary electron donor, vibrational spectroscopy
Abstract
The photooxidation of the primary electron donor in several Photosystem I-related organisms (Synechocystis sp. PCC 6803, Heliobacillus mobilis, and Chlorobium limicola f. sp. thiosulphatophilum) has been studied by lightinduced FTIR difference spectroscopy at 100 K in the 4000 to 1200 cm -1 spectral range. The data are compared to the well-characterized FTIR difference spectra of the photooxidation of the primary donor P in Rhodobacter sphaeroides (both wild type and the heterodimer mutant HL M202) in order to get information on the charge localization and the extent of coupling within the (bacterio)chlorophylls constituting the oxidized primary donors. In Rb. sphaeroides RC, four marker bands mostly related to the dimeric nature of the oxidized primary donor have been previously observed at ~ 2600, 1550, 1480, and 1295 cm - l . The high-frequency band has been shown to correspond to an electronic transition (Breton et al. (1992) Biochemistry 31: 7503-7510) while the three other marker bands have been described as phase-phonon bands (Reimers and Hush (1995) Chem Phys 197: 323-332). The absence of these bands in PS I as well as in the heterodimer HL M202 demonstrates that in P700 + the charge is essentially localized on a single chlorophyll molecule. For both H. mobilis and C. limicola, the presence of a high-frequency band at ~ 2050 and 2450 cm - l , respectively, and of phase-phonon bands (at ~ 1535 and 1300 cm -1 in H. mobilis, at .-~ 1465 and 1280 cm -1 in C. limicola) indicate that the positive charge in the photooxidized primary donor is shared between two coupled BChls. The structure of P840 + in C. limicola, in terms of the resonance interactions between the two BChl a molecules constituting the oxidized primary donor, is close to that of P+ in purple bacteria reaction centers while for H. mobilis the FTIR data are interpreted in terms of a weaker coupling of the two bacteriochlorophylls.
Abbreviations: (B)Chl-(bacterio)chlorophyll; BPhe-bacteriopheophytin; C.-Chlorobium; F T I R - F o u r i e r transform infrared; H.-Heliobacillus; PS I, PS II-Photosystem I, Photosystem II; P - p r i m a r y electron donor; R C - reaction center; Rb. -Rhodobacter; Rp. -Rhodopseudomonas; QA -primary quinone acceptor; W t - wild type Introduction
The reaction center core of Photosystem I (PS I) is a heterodimeric protein complex of two large polypeptides (82-83 kDa) that bind the primary electron donor
P700, the intermediate electron carriers A0, Al, and the 4Fe-4S iron-sulfur center Fx (Golbeck 1993). While there are several common functional and structural properties between the well-characterized reaction center (RC) of purple bacteria and the Photosys-
302 tem II from plants (Michel and Deisenhofer 1988), the RC of P S I is more related to that of green sulfur bacteria and of heliobacteria as they all contain ironsulfur clusters as terminal electron acceptors (Golbeck 1993). Several observations indicate that the RC core of heliobacteria and green sulfur bacteria is homodimeric, i.e. it is made of two identical protein subunits that bind the pigments (Liebl et al. 1993; Btittner et al. 1992; Oh-oka et al. 1995), in contrast to the heterodimeric structure of PS I, PS II, and purple bacteria RCs. In the RC of Heliobacillus (H.) mobilis, the primary electron donor P798 is presumably a dimer of bacteriochlorophyll g (BChl g) while in the green sulfur bacteria Chlorobium (C.) limicola, the primary donor P840 appears to be a dimer of BChl a (for reviews, see Amesz 1995 and Feiler and Hauska 1995). The primary electron donor of Photosystem I, P700, is probably a dimer of chlorophyll a (Chl a) although several optical studies have suggested that the positive charge in the P700 + radical cation is localized on only one Chl (reviewed in S6tif 1992; Evans and Nugent 1993). The electron density map at 4.5 ,~ resolution of PS I from Synechococcus sp. shows the contours of two Chls attributed to P700 (Krauss et al. 1993; Schubert et al. 1995). These two Chls appear much further apart compared to the distance between the two BChl macrocycles of the primary electron donor (P) in the RC of purple bacteria (Lancaster et al. 1995). Recent ENDOR and ESEEM studies of P700 + in P S I preparations from various species suggest that P700 + could be a strongly asymmetric dimer in which the unpaired electron is predominantly localized on one Chl molecule (Davis et al. 1993; K~iss et al. 1994; Cui et al. 1995). Furthermore, while low-temperature EPR studies of the triplet state of P700 (3P700) have indicated that the triplet resides on a monomeric Chl, timeresolved EPR measurements suggest that the triplet is delocalized over the two halves of a Chl dimer at room temperature (Sieckmann et al. 1993). It therefore appears that the nature of P700, P700 +, and 3P700 is still not fully understood. In the past few years, FTIR difference spectroscopy has become an increasingly important method for investigating photosynthetic RCs from bacteria and plants (for reviews, see Mantele 1993 and Nabedryk 1996). This approach which is highly sensitive to small alterations in bond lengths and angles has emerged as a powerful tool to investigate in detail the structural changes localized to individual bonds of the cofactors and the protein. Notably, in the light-induced (reviewed in Nabedryk 1996) and redox-
induced (Leonhard and M~intele 1990) FTIR difference spectra of Rhodobacter (Rb.) sphaeroides and Rhodopseudomonas (Rp.) viridis RCs corresponding to the photooxidation of the primary electron donor, bands have been assigned to C=O modes from the BChl dimer. In addition, a broad absorption band in the mid-IR centered near 2600 c m - l in Rb. sphaeroides and near 2750 c m - l in Rp. viridis has been shown to correspond to an electronic transition of the photooxidized primary donor, reflecting the magnitude of the resonance interactions between the two BChls PL and PM of P+ (Breton et al. 1992; Parson et al. 1992). An asymmetry of the charge distribution in P+ was needed to explain this mid-IR electronic transition. Furthermore, IR marker bands mostly related to the BChl dimeric nature of the primary donor in the oxidized state have been observed at ~ 1550, 1480, and 1295 cm -1 (reviewed in Nabedryk 1996). A correlation has long been noted between the 2600 or 2750 c m - 1 transition and the presence of the three positive IR marker bands in the P + Q - / P Q spectra of native RC from purple bacteria, e.g. they are altogether absent in the spectra of the two heterodimer mutants in which His M202 or His L173 was replaced by Leu (Nabedryk et al. 1992; Breton et al. 1992; Davis et al. 1992), or they are similarly affected by mutations introducing hydrogen bonds to the keto carbonyls of P (Nabedryk et al. 1993). Recently, these positive bands at ~ 1550, 1480, and 1295 cm -1 have been described as phase-phonon bands (Reimers and Hush 1995) reflecting transient deformation of the porphyrin ring when the hole is exchanging between the two halves of the dimer. Comparable bands around 1530, 1460, and 1260 c m - 1 have been observed to develop with a ~ 200 fs time constant following the formation of the electronically excited state P*, a process that is thought to reflect early charge separation within the special pair (Hamm and Zinth 1995). The photooxidation of P700 has been previously investigated at ambient temperature in the 1800 to 1200 cm -1 spectral range in pea thylakoids and the corresponding PS I-enriched particles (Tavitian et al. 1986), in thylakoids of the cyanobacterium Spirulina geitleiri (Tavitian 1987; Nabedryk et al. 1990a) as well as at - 10 °C on a P S I particle from the cyanobacterium Synechocystis (MacDonald et al. 1993). The data obtained from these different P S I particles are in good agreement. Comparison with model compound spectra of cation of Chl a and pyroChl a has notably eliminated the possibility that an enol could be formed upon photooxidation of P700 (Nabedryk et al. 1990a). In
303 I
the present work, we report on a light-induced FTIR analysis in the whole 4000 to 1200 cm -1 spectral range, at 100 K, of the photooxidation of the primary electron donor in PS I from Synechocystis and in the two PS 1-related organisms H. mobilis and C. limicola. These data are compared to the well-characterized FTIR difference spectra of the photooxidation of P in Rb. sphaeroides (both wild type and the heterodimer mutant HL M202) in order to get information on the charge localization and the extent of coupling within the (B)Chls constituting the oxidized primary donor.
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Material and methods
IR samples were prepared by drying under argon on CaF2 windows a suspension of either P S I particles from Synechocystis, or RC from Rb. sphaeroides wild type (Wt), or chromatophores from the heterodimer mutant HL M202 of Rb. sphaeroides, or chlorosomedepleted membranes from C. limicola (Albouy 1995), or membranes from H. mobilis. For P S I particles and membranes from C. limicola and H. mobilis, ascorbate was added to the suspension before drying in order to maximize the amount of reduced primary donor prior to illumination. Films were rehydrated as described in Breton et al. (1992) and cooled in the dark in a temperature-regulated cryostat. FTIR measurements were performed with a Nicolet 60 SX spectrometer equipped with a MCT-A detector. Light-minus-dark FTIR difference spectra were obtained under steady-state illumination conditions as previously described (Nabedryk et al. 1990b; Breton et al. 1992), using different filters (Schott), RG630 for PS I and RG715 for Rb. sphaeroides, C. limicola, and H. mobilis. Cycles of illumination were repeated several hundred times.
Results and discussion
Figure 1 shows the light-induced FTIR difference spectra in the 4000-1200 cm - l range for Rb. sphaeroides RC from wild type (a), Rb. sphaeroides chromatophores from the heterodimer mutant HL M202 (b), P S I particles from Synechocystis (c), membranes from H. mobilis (e) and C. limicola (f). The redox-induced FTIR difference spectrum (cationminus-neutral) of Chl a in tetrahydrofuran is shown for comparison in Figure ld (see also Nabedryk et al. 1990a). For Rb. sphaeroides RC and chromatophores,
FTIR spectroscopy of primary donor photooxidation in Photosystem I, Heliobacillus mobUis, and Chlorobium limicola. Comparison with purple bacteria Eliane Nabedryk, Winfried Leibl & Jacques Breton Section de Bio~nerg~tique, D~partement de Biologie Cellulaire et Moldculaire, CEA/Saclay, 91191 Gif-sur-Yvette Cedex, France Received 6 December 1995; accepted in revised form 12 February 1996
Key words: bacteriochlorophyll dimer, photosynthetic reaction center, primary electron donor, vibrational spectroscopy
Abstract
The photooxidation of the primary electron donor in several Photosystem I-related organisms (Synechocystis sp. PCC 6803, Heliobacillus mobilis, and Chlorobium limicola f. sp. thiosulphatophilum) has been studied by lightinduced FTIR difference spectroscopy at 100 K in the 4000 to 1200 cm -1 spectral range. The data are compared to the well-characterized FTIR difference spectra of the photooxidation of the primary donor P in Rhodobacter sphaeroides (both wild type and the heterodimer mutant HL M202) in order to get information on the charge localization and the extent of coupling within the (bacterio)chlorophylls constituting the oxidized primary donors. In Rb. sphaeroides RC, four marker bands mostly related to the dimeric nature of the oxidized primary donor have been previously observed at ~ 2600, 1550, 1480, and 1295 cm - l . The high-frequency band has been shown to correspond to an electronic transition (Breton et al. (1992) Biochemistry 31: 7503-7510) while the three other marker bands have been described as phase-phonon bands (Reimers and Hush (1995) Chem Phys 197: 323-332). The absence of these bands in PS I as well as in the heterodimer HL M202 demonstrates that in P700 + the charge is essentially localized on a single chlorophyll molecule. For both H. mobilis and C. limicola, the presence of a high-frequency band at ~ 2050 and 2450 cm - l , respectively, and of phase-phonon bands (at ~ 1535 and 1300 cm -1 in H. mobilis, at .-~ 1465 and 1280 cm -1 in C. limicola) indicate that the positive charge in the photooxidized primary donor is shared between two coupled BChls. The structure of P840 + in C. limicola, in terms of the resonance interactions between the two BChl a molecules constituting the oxidized primary donor, is close to that of P+ in purple bacteria reaction centers while for H. mobilis the FTIR data are interpreted in terms of a weaker coupling of the two bacteriochlorophylls.
Abbreviations: (B)Chl-(bacterio)chlorophyll; BPhe-bacteriopheophytin; C.-Chlorobium; F T I R - F o u r i e r transform infrared; H.-Heliobacillus; PS I, PS II-Photosystem I, Photosystem II; P - p r i m a r y electron donor; R C - reaction center; Rb. -Rhodobacter; Rp. -Rhodopseudomonas; QA -primary quinone acceptor; W t - wild type Introduction
The reaction center core of Photosystem I (PS I) is a heterodimeric protein complex of two large polypeptides (82-83 kDa) that bind the primary electron donor
P700, the intermediate electron carriers A0, Al, and the 4Fe-4S iron-sulfur center Fx (Golbeck 1993). While there are several common functional and structural properties between the well-characterized reaction center (RC) of purple bacteria and the Photosys-
302 tem II from plants (Michel and Deisenhofer 1988), the RC of P S I is more related to that of green sulfur bacteria and of heliobacteria as they all contain ironsulfur clusters as terminal electron acceptors (Golbeck 1993). Several observations indicate that the RC core of heliobacteria and green sulfur bacteria is homodimeric, i.e. it is made of two identical protein subunits that bind the pigments (Liebl et al. 1993; Btittner et al. 1992; Oh-oka et al. 1995), in contrast to the heterodimeric structure of PS I, PS II, and purple bacteria RCs. In the RC of Heliobacillus (H.) mobilis, the primary electron donor P798 is presumably a dimer of bacteriochlorophyll g (BChl g) while in the green sulfur bacteria Chlorobium (C.) limicola, the primary donor P840 appears to be a dimer of BChl a (for reviews, see Amesz 1995 and Feiler and Hauska 1995). The primary electron donor of Photosystem I, P700, is probably a dimer of chlorophyll a (Chl a) although several optical studies have suggested that the positive charge in the P700 + radical cation is localized on only one Chl (reviewed in S6tif 1992; Evans and Nugent 1993). The electron density map at 4.5 ,~ resolution of PS I from Synechococcus sp. shows the contours of two Chls attributed to P700 (Krauss et al. 1993; Schubert et al. 1995). These two Chls appear much further apart compared to the distance between the two BChl macrocycles of the primary electron donor (P) in the RC of purple bacteria (Lancaster et al. 1995). Recent ENDOR and ESEEM studies of P700 + in P S I preparations from various species suggest that P700 + could be a strongly asymmetric dimer in which the unpaired electron is predominantly localized on one Chl molecule (Davis et al. 1993; K~iss et al. 1994; Cui et al. 1995). Furthermore, while low-temperature EPR studies of the triplet state of P700 (3P700) have indicated that the triplet resides on a monomeric Chl, timeresolved EPR measurements suggest that the triplet is delocalized over the two halves of a Chl dimer at room temperature (Sieckmann et al. 1993). It therefore appears that the nature of P700, P700 +, and 3P700 is still not fully understood. In the past few years, FTIR difference spectroscopy has become an increasingly important method for investigating photosynthetic RCs from bacteria and plants (for reviews, see Mantele 1993 and Nabedryk 1996). This approach which is highly sensitive to small alterations in bond lengths and angles has emerged as a powerful tool to investigate in detail the structural changes localized to individual bonds of the cofactors and the protein. Notably, in the light-induced (reviewed in Nabedryk 1996) and redox-
induced (Leonhard and M~intele 1990) FTIR difference spectra of Rhodobacter (Rb.) sphaeroides and Rhodopseudomonas (Rp.) viridis RCs corresponding to the photooxidation of the primary electron donor, bands have been assigned to C=O modes from the BChl dimer. In addition, a broad absorption band in the mid-IR centered near 2600 c m - l in Rb. sphaeroides and near 2750 c m - l in Rp. viridis has been shown to correspond to an electronic transition of the photooxidized primary donor, reflecting the magnitude of the resonance interactions between the two BChls PL and PM of P+ (Breton et al. 1992; Parson et al. 1992). An asymmetry of the charge distribution in P+ was needed to explain this mid-IR electronic transition. Furthermore, IR marker bands mostly related to the BChl dimeric nature of the primary donor in the oxidized state have been observed at ~ 1550, 1480, and 1295 cm -1 (reviewed in Nabedryk 1996). A correlation has long been noted between the 2600 or 2750 c m - 1 transition and the presence of the three positive IR marker bands in the P + Q - / P Q spectra of native RC from purple bacteria, e.g. they are altogether absent in the spectra of the two heterodimer mutants in which His M202 or His L173 was replaced by Leu (Nabedryk et al. 1992; Breton et al. 1992; Davis et al. 1992), or they are similarly affected by mutations introducing hydrogen bonds to the keto carbonyls of P (Nabedryk et al. 1993). Recently, these positive bands at ~ 1550, 1480, and 1295 cm -1 have been described as phase-phonon bands (Reimers and Hush 1995) reflecting transient deformation of the porphyrin ring when the hole is exchanging between the two halves of the dimer. Comparable bands around 1530, 1460, and 1260 c m - 1 have been observed to develop with a ~ 200 fs time constant following the formation of the electronically excited state P*, a process that is thought to reflect early charge separation within the special pair (Hamm and Zinth 1995). The photooxidation of P700 has been previously investigated at ambient temperature in the 1800 to 1200 cm -1 spectral range in pea thylakoids and the corresponding PS I-enriched particles (Tavitian et al. 1986), in thylakoids of the cyanobacterium Spirulina geitleiri (Tavitian 1987; Nabedryk et al. 1990a) as well as at - 10 °C on a P S I particle from the cyanobacterium Synechocystis (MacDonald et al. 1993). The data obtained from these different P S I particles are in good agreement. Comparison with model compound spectra of cation of Chl a and pyroChl a has notably eliminated the possibility that an enol could be formed upon photooxidation of P700 (Nabedryk et al. 1990a). In
303 I
the present work, we report on a light-induced FTIR analysis in the whole 4000 to 1200 cm -1 spectral range, at 100 K, of the photooxidation of the primary electron donor in PS I from Synechocystis and in the two PS 1-related organisms H. mobilis and C. limicola. These data are compared to the well-characterized FTIR difference spectra of the photooxidation of P in Rb. sphaeroides (both wild type and the heterodimer mutant HL M202) in order to get information on the charge localization and the extent of coupling within the (B)Chls constituting the oxidized primary donor.
'
I
I
'
b
o
Material and methods
IR samples were prepared by drying under argon on CaF2 windows a suspension of either P S I particles from Synechocystis, or RC from Rb. sphaeroides wild type (Wt), or chromatophores from the heterodimer mutant HL M202 of Rb. sphaeroides, or chlorosomedepleted membranes from C. limicola (Albouy 1995), or membranes from H. mobilis. For P S I particles and membranes from C. limicola and H. mobilis, ascorbate was added to the suspension before drying in order to maximize the amount of reduced primary donor prior to illumination. Films were rehydrated as described in Breton et al. (1992) and cooled in the dark in a temperature-regulated cryostat. FTIR measurements were performed with a Nicolet 60 SX spectrometer equipped with a MCT-A detector. Light-minus-dark FTIR difference spectra were obtained under steady-state illumination conditions as previously described (Nabedryk et al. 1990b; Breton et al. 1992), using different filters (Schott), RG630 for PS I and RG715 for Rb. sphaeroides, C. limicola, and H. mobilis. Cycles of illumination were repeated several hundred times.
Results and discussion
Figure 1 shows the light-induced FTIR difference spectra in the 4000-1200 cm - l range for Rb. sphaeroides RC from wild type (a), Rb. sphaeroides chromatophores from the heterodimer mutant HL M202 (b), P S I particles from Synechocystis (c), membranes from H. mobilis (e) and C. limicola (f). The redox-induced FTIR difference spectrum (cationminus-neutral) of Chl a in tetrahydrofuran is shown for comparison in Figure ld (see also Nabedryk et al. 1990a). For Rb. sphaeroides RC and chromatophores,
