PhotosynthesisResearch 47: 41-49, 1996. (~) 1996KluwerAcademicPublishers. Printedin the Netherlands. Regular paper
Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer Szabolcs Osv~ith 1, G~ibor Laczk61, Pierre Sebban 2 & P6ter Mar6ti 1 1Department of Biophysics, Jdzsef Attila University, Szeged, Egyetem utca 2, H-6722, Hungary; 2Centre de G~n~tique Mol~culaire CNRS, Gif-sur-Yvette, Avenue de la Terrasse, Bat. 24, F-91198, France Received29 June 1995.acceptedin revisedform20 October1995
Key words: bacterial photosynthesis, fluorescence induction, reaction center protein
Abstract
Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15 -4- 0.1) than in Rhodobacter capsulatus (1.2 -t- 0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary qninone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.
Abbreviations: Bchl-bacteriochlorophyll; Bpheo-bacteriopheophytin; D-electron donor to P+; P bacteriochlorophyU dimer; Q - quinone acceptor; QA -primary quinone acceptor; QB - secondary quinone acceptor; R C - reaction center protein; UQ6 - ubiquinone-30 Introduction
The primary photochemical events of photosynthesis occur in a specially organized membrane-bound pigment-protein complex, called reaction center (RC). The RCs from non-sulphur purple bacteria have long been well suited for structural and functional studies as their crystal structure is known for several strains with high spatial resolution and the electron and proton transfers in these RCs are relatively simple as com-
pared to those in higher plants (Clayton 1980; Feher et al. 1989; Ermler et al. 1994; Deisenhofer et al. 1995). The photon absorbed by the bacterioehlorophyll dimer (P) initiates charge separation with a quantum yield of near unity (Wraight and Clayton 1974). The separated charges are stabilized by subsequent intermediate redox states. P transfers its electron in about 200 ps to the first stable electron acceptor (QA) through monomeric bacteriochlorophyll and bacteriopheophytin. The primary quinone is oxidized in
42 20...200 #s (depending on the species) by the secondary quinone (Qa). If P+ is re-reduced by an electron donor (D) before charge recombination but after interquinone electron transfer, the RC will be ready for additional charge separation: kx ~ kD DPQAQe
...... < ......
~
DP*QA'Qa
~ ....
>
DP*QAQe-
---->
D*PQAQa"
kpA
Scheme 1 Here, ki, kpA and kD are the rates of photochemistry (charge separation), thermal back reaction from state P+QA and re-reduction of P+ by external electron donor, respectively and Ke denotes the electron equilibrium constant in the acceptor quinone complex. QB functions as two-electron acceptor: after reduction with two electrons and two protons, QBH2 is released from the RC and is replaced by an oxidized quinone from the quinone pool. The turnover time of the photochemical cycle is about 1 ms. The majority of flash-induced electron transport processes in bacterial RC have routinely been followed by time-resolved absorption spectrophotometry. Bacteriochlorophyll fluorescence either from the RC (Clayton 1972, 1980; Schenck et al. 1981) or from the antenna (Godik and Borisov 1977; Amesz and Vasmel 1986; Deinum et al. 1991; Hoff and Fischer 1993) can also be used to monitor redox changes in the RC. The direct relation between the fluorescence yield and the redox state of the reaction center was first demonstrated using purple photosynthetic bacterium RhodospiriUum rubrum (Vredenberg and Duysens 1963). It was found that the light-induced bleaching of dimer in the RC blocked the subsequent excitation for photosynthesis and thus the probability of loss of the excitation energy by antenna-fluorescence increased. The relatively large changes in Bchl fluorescence yield of photosynthetic bacteria have been used mainly to study energy transfer and trapping (for a recent review, see Van Grondelle et al. 1994) but rarely as a probe for electron transfer in the RC. Upon excitation, the dimer in the RC of Rhodobacter (Rb.) sphaeroides emits fluorescence in the near infrared spectral range (centered around 910 nm) (Zankel et al. 1968; Clayton 1977; Stadnichuk and Lukashev 1982). The quantum yield of dimer fluorescence is very low (4.10 -4 ) as compared to that of light-harvesting Bchl measured in chromatophores or whole cells (2.10 -2 to 1.10-1 depending on the strain) (Clayton 1977, 1980). The low fluorescence yield of the dimer in the RC shows that the excitation reach-
ing this pigment is quenched strongly due mainly to the high quantum efficiency of the photochemistry (charge separation) (Zankel et al. 1968). Additional inconvenience of the method based on dimer fluorescence is the lack of fast electron donor to P+ in isolated RC of Rb. sphaeroides and Rb. capsulatus. As the dimer remains oxidized for a long period of time and P+ is not a precursor of dimer fluorescence, low level of fluorescence will be measured independently of the redox state of the acceptor complex. It was shown that the yield of fluorescence at 920 nm increased by a factor of about three upon closing the RC (PQA --rPQA ) either chemically (using sodium dithionite) or photochemically (by illumination in the presence of an electron donor) (Clayton 1966). Here, we set the aim to determine the origin and the relative yield of observed fluorescence in different redox states of the RC isolated from strains Rb. sphaeroides and Rb. capsulatus which show high degree of sequence homology (79% for the quinone binding sites) (see Komiya et al. 1988). The fluorescence from the dimer is used to track forward (rereduction of P+ by cytochrome and reduction of Qa by QA) and backward (charge recombination) electron transfer reactions. Our results demonstrate the effectiveness of dimer fluorescence as a supplementary method to absorption change techniques in study of electron transfer reactions in RCs at room temperature.
Materials and methods
Reaction centers from the wild (2.4.1.) and carotenoidless mutant (R-26) of the strain Rb. sphaeroides and from Rb. capsulatus were prepared as described earlier (Mar6ti and Wraight 1988; Baciou et al. 1993). The RC preparation was purified by repeated DEAE sepharose column chromatography until the purity (OD(280 nm)/OD(800 nm)) dropped below 1.30 and 1.50 in Rb. sphaeroides and Rb. capsulatus, respectively. The secondary quinone activity of the RC was reconstituted by UQ6 (Sigma). Ferrocene and reduced cytochrome c were used as external electron donor to p+. Light-induced absorption changes and fluorescence of the RC were measured simultaneously. Fluorescence was detected through the bottom side of a 1 x 1 x 5 cm four-sided rectangular cuvette by a redsensitive photomultiplier (Hamamatsu Type R331003) and digitalized using a Philips PM 3350A digital
43 storage oscilloscope connected to an IBM AT compatible PC. The photomultiplier was protected from the exciting light by a red cut-off filter (850 nm) and a 915 nm interference filter of approximately 15 nm bandwidth. The fluorescence was excited either by a laser diode (Laser Diode, Inc. Type LCW-100, emission wavelength 810 nm, power 500 mW) or by a short (tl/2 ~ 6 /~s) Xe-flash (General Radio, Stroboslave Type 1539-A). Due to the very short rise and fall times (100 ns) of the laser diode, the pulsed illumination by laser diode could be considered as 'rectangular shape of excitation' in our time frame. Expansion of the exciting beam resulted in a homogeneous excitation which was crucial to get true (not mixed) kinetics. The directions of the excitation, the observation of fluorescence and the monitoring beam for absorption measurements were mutually perpendicular. The charge recombination and the semiquinone oscillation were monitored by absorption changes at 865 nm and 450 nm, respectively. The spectral changes of the kinetic traces of fluorescence upon rectangular shape of excitation at 810 nm were measured through a grating monochromator (Jobin-Yvon Model H20 IR) in a home-built fluorometer. The sensitivity of the fluorometer as a function of wavelength was determined by recording the 2850 K black body radiation of a tungsten lamp (Osram). Any contribution of scattered exciting light in the emission spectra and kinetic changes at 915 nm was found to be negligible. Excitation spectra for flash-induced fluorescence at 915 nm were corrected for variations with wavelength in emission of a Xe flash lamp (EG&G FX200), grating transmission of the monochromator and photodetector response. The spectral resolution was 4 nm. Excitation spectra for 915 nm fluorescence were taken in two different (PQA and P+ QA) states of the RC. As the actinic effect of Xe flash passed through the monochromator was negligible, a saturating laser diode illumination (1 ms) prior to the Xe flash could switch the redox state of RC from PQA to P+Q~. Thus, the use of chemicals as redox agents with their obscure effects on fluorescence yield of RC and (contaminating) pigments (see Clayton 1977) could be avoided.
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Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer Szabolcs Osv~ith 1, G~ibor Laczk61, Pierre Sebban 2 & P6ter Mar6ti 1 1Department of Biophysics, Jdzsef Attila University, Szeged, Egyetem utca 2, H-6722, Hungary; 2Centre de G~n~tique Mol~culaire CNRS, Gif-sur-Yvette, Avenue de la Terrasse, Bat. 24, F-91198, France Received29 June 1995.acceptedin revisedform20 October1995
Key words: bacterial photosynthesis, fluorescence induction, reaction center protein
Abstract
Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15 -4- 0.1) than in Rhodobacter capsulatus (1.2 -t- 0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary qninone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.
Abbreviations: Bchl-bacteriochlorophyll; Bpheo-bacteriopheophytin; D-electron donor to P+; P bacteriochlorophyU dimer; Q - quinone acceptor; QA -primary quinone acceptor; QB - secondary quinone acceptor; R C - reaction center protein; UQ6 - ubiquinone-30 Introduction
The primary photochemical events of photosynthesis occur in a specially organized membrane-bound pigment-protein complex, called reaction center (RC). The RCs from non-sulphur purple bacteria have long been well suited for structural and functional studies as their crystal structure is known for several strains with high spatial resolution and the electron and proton transfers in these RCs are relatively simple as com-
pared to those in higher plants (Clayton 1980; Feher et al. 1989; Ermler et al. 1994; Deisenhofer et al. 1995). The photon absorbed by the bacterioehlorophyll dimer (P) initiates charge separation with a quantum yield of near unity (Wraight and Clayton 1974). The separated charges are stabilized by subsequent intermediate redox states. P transfers its electron in about 200 ps to the first stable electron acceptor (QA) through monomeric bacteriochlorophyll and bacteriopheophytin. The primary quinone is oxidized in
42 20...200 #s (depending on the species) by the secondary quinone (Qa). If P+ is re-reduced by an electron donor (D) before charge recombination but after interquinone electron transfer, the RC will be ready for additional charge separation: kx ~ kD DPQAQe
...... < ......
~
DP*QA'Qa
~ ....
>
DP*QAQe-
---->
D*PQAQa"
kpA
Scheme 1 Here, ki, kpA and kD are the rates of photochemistry (charge separation), thermal back reaction from state P+QA and re-reduction of P+ by external electron donor, respectively and Ke denotes the electron equilibrium constant in the acceptor quinone complex. QB functions as two-electron acceptor: after reduction with two electrons and two protons, QBH2 is released from the RC and is replaced by an oxidized quinone from the quinone pool. The turnover time of the photochemical cycle is about 1 ms. The majority of flash-induced electron transport processes in bacterial RC have routinely been followed by time-resolved absorption spectrophotometry. Bacteriochlorophyll fluorescence either from the RC (Clayton 1972, 1980; Schenck et al. 1981) or from the antenna (Godik and Borisov 1977; Amesz and Vasmel 1986; Deinum et al. 1991; Hoff and Fischer 1993) can also be used to monitor redox changes in the RC. The direct relation between the fluorescence yield and the redox state of the reaction center was first demonstrated using purple photosynthetic bacterium RhodospiriUum rubrum (Vredenberg and Duysens 1963). It was found that the light-induced bleaching of dimer in the RC blocked the subsequent excitation for photosynthesis and thus the probability of loss of the excitation energy by antenna-fluorescence increased. The relatively large changes in Bchl fluorescence yield of photosynthetic bacteria have been used mainly to study energy transfer and trapping (for a recent review, see Van Grondelle et al. 1994) but rarely as a probe for electron transfer in the RC. Upon excitation, the dimer in the RC of Rhodobacter (Rb.) sphaeroides emits fluorescence in the near infrared spectral range (centered around 910 nm) (Zankel et al. 1968; Clayton 1977; Stadnichuk and Lukashev 1982). The quantum yield of dimer fluorescence is very low (4.10 -4 ) as compared to that of light-harvesting Bchl measured in chromatophores or whole cells (2.10 -2 to 1.10-1 depending on the strain) (Clayton 1977, 1980). The low fluorescence yield of the dimer in the RC shows that the excitation reach-
ing this pigment is quenched strongly due mainly to the high quantum efficiency of the photochemistry (charge separation) (Zankel et al. 1968). Additional inconvenience of the method based on dimer fluorescence is the lack of fast electron donor to P+ in isolated RC of Rb. sphaeroides and Rb. capsulatus. As the dimer remains oxidized for a long period of time and P+ is not a precursor of dimer fluorescence, low level of fluorescence will be measured independently of the redox state of the acceptor complex. It was shown that the yield of fluorescence at 920 nm increased by a factor of about three upon closing the RC (PQA --rPQA ) either chemically (using sodium dithionite) or photochemically (by illumination in the presence of an electron donor) (Clayton 1966). Here, we set the aim to determine the origin and the relative yield of observed fluorescence in different redox states of the RC isolated from strains Rb. sphaeroides and Rb. capsulatus which show high degree of sequence homology (79% for the quinone binding sites) (see Komiya et al. 1988). The fluorescence from the dimer is used to track forward (rereduction of P+ by cytochrome and reduction of Qa by QA) and backward (charge recombination) electron transfer reactions. Our results demonstrate the effectiveness of dimer fluorescence as a supplementary method to absorption change techniques in study of electron transfer reactions in RCs at room temperature.
Materials and methods
Reaction centers from the wild (2.4.1.) and carotenoidless mutant (R-26) of the strain Rb. sphaeroides and from Rb. capsulatus were prepared as described earlier (Mar6ti and Wraight 1988; Baciou et al. 1993). The RC preparation was purified by repeated DEAE sepharose column chromatography until the purity (OD(280 nm)/OD(800 nm)) dropped below 1.30 and 1.50 in Rb. sphaeroides and Rb. capsulatus, respectively. The secondary quinone activity of the RC was reconstituted by UQ6 (Sigma). Ferrocene and reduced cytochrome c were used as external electron donor to p+. Light-induced absorption changes and fluorescence of the RC were measured simultaneously. Fluorescence was detected through the bottom side of a 1 x 1 x 5 cm four-sided rectangular cuvette by a redsensitive photomultiplier (Hamamatsu Type R331003) and digitalized using a Philips PM 3350A digital
43 storage oscilloscope connected to an IBM AT compatible PC. The photomultiplier was protected from the exciting light by a red cut-off filter (850 nm) and a 915 nm interference filter of approximately 15 nm bandwidth. The fluorescence was excited either by a laser diode (Laser Diode, Inc. Type LCW-100, emission wavelength 810 nm, power 500 mW) or by a short (tl/2 ~ 6 /~s) Xe-flash (General Radio, Stroboslave Type 1539-A). Due to the very short rise and fall times (100 ns) of the laser diode, the pulsed illumination by laser diode could be considered as 'rectangular shape of excitation' in our time frame. Expansion of the exciting beam resulted in a homogeneous excitation which was crucial to get true (not mixed) kinetics. The directions of the excitation, the observation of fluorescence and the monitoring beam for absorption measurements were mutually perpendicular. The charge recombination and the semiquinone oscillation were monitored by absorption changes at 865 nm and 450 nm, respectively. The spectral changes of the kinetic traces of fluorescence upon rectangular shape of excitation at 810 nm were measured through a grating monochromator (Jobin-Yvon Model H20 IR) in a home-built fluorometer. The sensitivity of the fluorometer as a function of wavelength was determined by recording the 2850 K black body radiation of a tungsten lamp (Osram). Any contribution of scattered exciting light in the emission spectra and kinetic changes at 915 nm was found to be negligible. Excitation spectra for flash-induced fluorescence at 915 nm were corrected for variations with wavelength in emission of a Xe flash lamp (EG&G FX200), grating transmission of the monochromator and photodetector response. The spectral resolution was 4 nm. Excitation spectra for 915 nm fluorescence were taken in two different (PQA and P+ QA) states of the RC. As the actinic effect of Xe flash passed through the monochromator was negligible, a saturating laser diode illumination (1 ms) prior to the Xe flash could switch the redox state of RC from PQA to P+Q~. Thus, the use of chemicals as redox agents with their obscure effects on fluorescence yield of RC and (contaminating) pigments (see Clayton 1977) could be avoided.
A •~,
2
8 '...~L=' '
I...........................
0
o12
0
o14
.
. . . .
o16
ole
T i m e (s) 1.2" B
.--
1
~
0.8
•
g -50
~ 0.6
100
0.4 .150
