PhotosynthesisResearch 45: 135-146, 1995. © 1995KluwerAcademicPublishers. Printedin the Netherlands. Regular paper
The Ile L229 --~ Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides Jfilia T a n d o r i , L~iszl6 N a g y , J~gnes Pusk~is 1, M a g d o l n a D r o p p a 1, G~ibor Horv~ith 1 & P 6 t e r Mar6ti*
J6zsef Attila University, Department of Biophysics, Szeged, Egyetem u. 2., H-6722, Hungary; 1Institute of Plant Biology, Biological Research Center, P.O.Box 521, Szeged, H-6701, Hungary; * Author for correspondence Received 27 September1994;acceptedin revisedform3 July 1995
This workis dedicatedto the memoryof Randall Ross Stein (1954-1994)and is, in a small way,a testamentto the impactwhichRandy'sideas have had on the developmentof the field of competitiveherbicidebinding.
Key words: bacterial photosynthesis, electron transfer, herbicide resistance
Abstract
A spontaneous mutant (RI89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 #M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF = 20) in UQo reconstituted isolated reaction centers and terbutryne in reaction centers (RF = 55) and in chromatophores (RF = 85). The amino acid sequence of the QB binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (IleL229 ~ Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone QB. The binding and redox properties of QB in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of QAQB- with respect to QA-QB was AGAB = --60 meV and 0 meV in reaction centers and AGAB = --85 meV and - 4 6 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQo and semiquinone UQo- in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of QB.
Abbreviations: BChl - bacteriochlorophyll; Ile - isoleucine; Met - methionin; P - primary donor; QA - primary quinone acceptor; QB - secondary quinone acceptor; RC - reaction center protein; UQo - 2,3-dimethoxy-5-methyl benzoquinone; UQ10 - ubiquinone 50 Introduction
The reaction center (RC) pigment-protein complex of photosynthetic bacteria is the minimum structural and functional unit which can catalyze electron and proton transfer processes on light excitation leading to stabilization of charges. The photon absorption by the bacteriochlorophyll dimer (primary donor, P) initiates electron transfer from P to the tightly bound prima-
ry quinone (QA) and subsequently, if available, to the more loosely bound secondary quinone (QB) (Feher et al. 1989). The acceptor quinone complex acts as a twoelectron gate: the reducing equivalents (electrons and protons) are exported in pairs (in form of hydroquinol, QBH2) from the RC. The released dihydroquinol is then replaced by a quinone from the pool resetting the system to the initial state (McPherson et al. 1990).
136 The X-ray diffraction studies of RC crystals of Rps. viridis (Michel et al. 1986) and Rb. sphaeroides (Allen et al. 1988; Ermler et al. 1994) have revealed the atomic structure of the QB binding domain. The carbonyl oxygens (0-2 and 0-5) of the head group of QB may form hydrogen bonds with N-61 atom of HisL19° and the side chain of SerL223, respectively. Similarly to the QA binding site, the QB pocket has highly hydrophobic internal residues but is surrounded by cluster of protonatable groups from the cytoplasmic phase (Gunner and Honig 1992). The structure and function of the acceptor quinone complex of the bacterial RC are highly similar to those of Photosystem II RC in higher plants (Shlnkarev and Wraight 1993; Barber and Andersson 1994). Based upon the knowledge about one system, this similarity has resulted in productive hypothesis of structure (Michel and Deisenhofer 1988), protonation (Lavergne and Junge 1993; Mar6ti 1993) or effect of bicarbonate (Wang et al. 1992) on the other. The bacterial systems are simpler and sometimes more amenable to study, but the plant systems are more widely investigated and of more commercial value. Thanks to the atomic resolution of the structure of bacterial RCs, the bacterial RC has served as an excellent model to understand the structural requirements of herbicide action in RCs (Paddock et al. 1988, 1991; Draber et al. 1991). Bacterial RCs are mainly sensitive to herbicides of triazine family (Stein et al. 1984) although recently mutants of the RCs of Rps. viridis (Sinning et al. 1989), Rb. capsulatus (Horv~th et al. 1991) and Rb. sphaeroides (Nagy et al. 1991) were found to be sensitive to diuron, an urea-type herbicide. Many of the commercially important herbicides act by competing with the quinone for the secondary quinone binding site both in green plant and bacterial systems (Wraight 1981; Stein et al. 1984). To investigate the fine details of the function of the acceptor quinone complex (including protonation, herbicide action, quinone binding affinity and the relation between the bacterial RC and PS II), specific residues in the vicinity of QB were mutated. That could be achieved by either site-directed mutagenesis (Bylina and Youvan 1987) or selection of herbicide resistant mutants (Sutton et al. 1984). As herbicides compete for the QB site, herbicide resistant mutants must involve amino acid residue changes in (or near to) the QB pocket. Similarities were recognized between sites of herbicide resistant mutations in the L subunit of bacterial RCs and D1 polypeptide in PS II: SerL223 and S e r 264
(Oettmeier 1992) and IleL229 and Leu 271 (Ohad and Hirschberg 1992), respectively. In this study electron transfer, quinone binding and inhibition measurements were carried out on spontaneous atrazin resistant mutant of Rhodobacter sphaeroides R-26 (R/89). The mutation was located in the L subunit near to the binding site for QB: IleL229 Met. This amino acid is not involved directly in the proton transfer to QB, but makes the QB binding stable being in van der Waals contact with it. The R/89 mutant is the carotenoidless equivalent of the mutant isolated earlier from Rhodobacter sphaeroides 2.4.1. by Paddock et al. 1988. Replacement of IleL229in other strains like Rb. capsulatus (Bylina and Youvan 1987; Baciou et al. 1993) and Rps. viridis (Sinning and Michel 1987) attracted special interest. The IleL229 ~ Ser mutation in Rb. capsulatus decreased the free energy of stabilization of the secondary semiquinone and lowered the binding affinity of UQ6 but did not cause resistance to inhibitors (Baciou et al. 1993). We determined the dissociation constants of water soluble quinone UQo, terbutryne and o-phenanthroline for the mutant R/89. The overlapping effects of altered electron transfer and thermodynamic properties of QB were separated from the changes in quinone binding affinity. It was observed that subtle structural changes in the mutant increased dramatically the dissociation constant of the quinone and, as a consequence, decreased the free energy of stabilization of the secondary semiquinone, QB-. The known structure of the native RC is used to deduce possible structural alterations in the mutant.
Materials and methods
Chemicals The buffers succinate, citrate, MES (2-[Nmorpholino]ethanesulfonic acid), MOPS (3-[Nmorpholino]propansulfonic acid), TRIS (2-amino-2(hydroxymethyl)-l,3-propanediol), BTP (1,3-bis(tris(hydroxymethyl)methylamino)-propane), CHES (2[N-cyclohexylamino] ethanesulfonic acid), CAPS (3(cyclohexylamino)-1-Propane-sulfonic acid) and horse heart ferricytochrome C were obtained from Sigma. Solutions of ferrocene and UQo (Sigma) were prepared in ethanol prior to use. Ubiquinone-50 (UQ10, 2,3-dimethoxy-5-methyl-6-decaisoprenyl-1,4benzoquinone, Sigma) was solubilized by sonication in
137 30% Triton X-100 (octylphenol-polyethyleneglycolether, Serva).
Strains and growth conditions Rhodobacter sphaeroides R-26 (R-26) strain was grown photoheterotrophically under anaerobic conditions in a medium supplemented with potassium succinate as described earlier (Ormerod et al. 1961). Spontaneous atrazin resistant R/89 strain was selected by transferring the wild type R-26 cells to the culture medium containing 200 #M atrazin. Cultures were then plated on to atrazin containing agar plates and incubated anaerobically in low light. Colonies developed on plates were used as inocula for photoheterotrophic growth in liquid medium. Growth temperature was 30 °C and the light intensity of ~ 90 #E m -2 s -1 was provided by tungsten lamps (Nagy et al. 1991). Cloning and sequencing the R/89 DNA p B S + / - DNA used for cloning and sequencing was prepared by an alkali lysis procedure (Maniatis et al. 1982). E. coli XL1-Blue (Bullock et al. 1987) was used as a host both for plasmid and phage vectors. Restriction endonucleases were obtained from New England BioLabs, proteinase K from Sigma and lysozyme from Reanal. ['7-32P]ATP was from Izinta, [a-35S] dATP and multiprime DNA labelling kit from Amersham. The hybridization probe was an ,-~ 850 bp XbaI-SalI fragment of a pUC 18 clone containing the R-26 pufL sequence except for the last 66 bases of the gene (a gift from Dr Mike Jones, University of Sheffield). An internal oligonucleotide primer with a sequence of Rb. sphaeroides 2.4.1.245L-9-51L non-coding strand (Williams et al. 1984) was synthesized in a Cyclone Plus (Milligene Biosearch) system. T7 DNA sequencing kit from Pharmacia and Sequenase version 2 from USB were used to determine the DNA sequence, and Microgenie (Beckmann) program was used for the analysis. Photoheterotrophically grown Rb. sphaeroides R/89 was harvested in early stationary phase. The genomic DNA was prepared following the method of Fornari and Kaplan (1983) and digested with PstI. XL1-Blue competent cells for cloning the 4-6 kb fraction were freshly prepared and transformed by the method of Chung et al. (1989). XL1-Blue frozen competent stock prepared by the same method was used for all the further transformation. Single standard DNA from pBS subclones was prepared by superinfection
by VCS M13 helper phage, and all the (single strand) templates were purified. The fragments resulted in the sequencing reactions were separated on 0.025 × 30 x 40 cm 5 or 6% denaturing polyacrylamide gels.
Preparation of chromatophores and reaction centers Cells were broken by French press ( ~ 150 MPa) and after removing unbroken cells and cell debris by centrifugation (20 min, ~ 40 000 × g) the chromatophores were sedimented by ultracentrifugation (90 min, 240 000 × g). Reaction centers were prepared by LDAO (lauryldimethylamine N-oxide, Fluka) solubilization and standard protein purification methods (ammonium sulphate precipitation, DEAE Sephacell (Sigma) column chromatography and ultrafiltration) as described previously (Mar6ti and Wraight 1988). RCs with one quinone were prepared as described by Okamura et al. (1975) with minor modifications. QB was reconstituted by adding a 10-fold excess of UQ10 to the RC solutions.
Optical measurements Flash-induced absorption changes were measured by a single-beam spectrophotometer of local design. The excitation was achieved by a xenon flash lamp (type EG&G FX200, tl/2 ~ 8.5 #s) passed through a 760 nm cut-off filter. The photomultiplier (EM19558) was protected by a Corning 4.96 blue filter. The analog signal of the photomultiplier was converted with an A/D converter card (Advantech PCL818) attached to an IBM-compatible 386 AT computer. The reconstitution of the secondary quinone activity of the RC was determined by decomposing the kinetic trace of the flash-induced absorption change due to the dark re-reduction of the oxidized primary donor P+ measured at 430 nm (Kleinfeld et al. 1984). The two-electron gate function of the acceptor quinone complex was investigated by two assays: semiquinone absorbance change measured at 450 nm (Vermeglio and Clayton 1977; Tandori et al. 1991) and cyt c 2+ oxidation detected at 550 nm (Kleinfeld et al. 1985) after trains of saturating flashes.
138 Results
46'
Cloning and sequencing The pulL containing PstI fragment appeared to be 4.5 kb by Southern hybridization, similarly to that found for Rb. sphaeroides 2.4.1. earlier (Williams et al. 1984). The PstI digested genomic DNA was separated on a 0.8% preparative agarose gel, and the electroeluated ,,~ 4-5 kb fraction was ligated into pBS+ linearized by PstI previously. Two of the screened 200 recombinants - resulted in the transformation of E. coli XL1-Blue with the ligation mixture above - were found to be positive by colony hybridization. However, only one of them contained the 4.5 kb PstI insert and gave positive signal, when purified DNA was analyzed by separation and hybridization in the agarose gel (Tsao et al. 1983). The 4.5 kb PstI insert was cut with PvuII, and both the 1.2 and 3.3 kb fragments were ligated into SmaI and PstI sites of pBS+. An ,-~ 1.7 kb SmaI PstI fragment of the 4.5 kb insert was subcloned similarly, and the entire sequence of the pufL was determined from the subclones. Comparing with the putL sequence published for the Rb. sphaeroides 2.4.1. the only difference was a C ~ G exchange on the noncoding strand resulted an IleL229 ---+Met point mutation. Since the same amino acid change has been published for Rb. sphaeroides 2.4.1. (Paddock et al. 1988), the R/89 seems to be its carotenoidless pair.
30' ~ 20' a 10' o
.
A
o.~
e-. c--
25
oe-
20
0
0:4
0'.s
1:2
116
2.0
B
ffl
-Q
The Ile L229 --~ Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides Jfilia T a n d o r i , L~iszl6 N a g y , J~gnes Pusk~is 1, M a g d o l n a D r o p p a 1, G~ibor Horv~ith 1 & P 6 t e r Mar6ti*
J6zsef Attila University, Department of Biophysics, Szeged, Egyetem u. 2., H-6722, Hungary; 1Institute of Plant Biology, Biological Research Center, P.O.Box 521, Szeged, H-6701, Hungary; * Author for correspondence Received 27 September1994;acceptedin revisedform3 July 1995
This workis dedicatedto the memoryof Randall Ross Stein (1954-1994)and is, in a small way,a testamentto the impactwhichRandy'sideas have had on the developmentof the field of competitiveherbicidebinding.
Key words: bacterial photosynthesis, electron transfer, herbicide resistance
Abstract
A spontaneous mutant (RI89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 #M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF = 20) in UQo reconstituted isolated reaction centers and terbutryne in reaction centers (RF = 55) and in chromatophores (RF = 85). The amino acid sequence of the QB binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (IleL229 ~ Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone QB. The binding and redox properties of QB in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of QAQB- with respect to QA-QB was AGAB = --60 meV and 0 meV in reaction centers and AGAB = --85 meV and - 4 6 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQo and semiquinone UQo- in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of QB.
Abbreviations: BChl - bacteriochlorophyll; Ile - isoleucine; Met - methionin; P - primary donor; QA - primary quinone acceptor; QB - secondary quinone acceptor; RC - reaction center protein; UQo - 2,3-dimethoxy-5-methyl benzoquinone; UQ10 - ubiquinone 50 Introduction
The reaction center (RC) pigment-protein complex of photosynthetic bacteria is the minimum structural and functional unit which can catalyze electron and proton transfer processes on light excitation leading to stabilization of charges. The photon absorption by the bacteriochlorophyll dimer (primary donor, P) initiates electron transfer from P to the tightly bound prima-
ry quinone (QA) and subsequently, if available, to the more loosely bound secondary quinone (QB) (Feher et al. 1989). The acceptor quinone complex acts as a twoelectron gate: the reducing equivalents (electrons and protons) are exported in pairs (in form of hydroquinol, QBH2) from the RC. The released dihydroquinol is then replaced by a quinone from the pool resetting the system to the initial state (McPherson et al. 1990).
136 The X-ray diffraction studies of RC crystals of Rps. viridis (Michel et al. 1986) and Rb. sphaeroides (Allen et al. 1988; Ermler et al. 1994) have revealed the atomic structure of the QB binding domain. The carbonyl oxygens (0-2 and 0-5) of the head group of QB may form hydrogen bonds with N-61 atom of HisL19° and the side chain of SerL223, respectively. Similarly to the QA binding site, the QB pocket has highly hydrophobic internal residues but is surrounded by cluster of protonatable groups from the cytoplasmic phase (Gunner and Honig 1992). The structure and function of the acceptor quinone complex of the bacterial RC are highly similar to those of Photosystem II RC in higher plants (Shlnkarev and Wraight 1993; Barber and Andersson 1994). Based upon the knowledge about one system, this similarity has resulted in productive hypothesis of structure (Michel and Deisenhofer 1988), protonation (Lavergne and Junge 1993; Mar6ti 1993) or effect of bicarbonate (Wang et al. 1992) on the other. The bacterial systems are simpler and sometimes more amenable to study, but the plant systems are more widely investigated and of more commercial value. Thanks to the atomic resolution of the structure of bacterial RCs, the bacterial RC has served as an excellent model to understand the structural requirements of herbicide action in RCs (Paddock et al. 1988, 1991; Draber et al. 1991). Bacterial RCs are mainly sensitive to herbicides of triazine family (Stein et al. 1984) although recently mutants of the RCs of Rps. viridis (Sinning et al. 1989), Rb. capsulatus (Horv~th et al. 1991) and Rb. sphaeroides (Nagy et al. 1991) were found to be sensitive to diuron, an urea-type herbicide. Many of the commercially important herbicides act by competing with the quinone for the secondary quinone binding site both in green plant and bacterial systems (Wraight 1981; Stein et al. 1984). To investigate the fine details of the function of the acceptor quinone complex (including protonation, herbicide action, quinone binding affinity and the relation between the bacterial RC and PS II), specific residues in the vicinity of QB were mutated. That could be achieved by either site-directed mutagenesis (Bylina and Youvan 1987) or selection of herbicide resistant mutants (Sutton et al. 1984). As herbicides compete for the QB site, herbicide resistant mutants must involve amino acid residue changes in (or near to) the QB pocket. Similarities were recognized between sites of herbicide resistant mutations in the L subunit of bacterial RCs and D1 polypeptide in PS II: SerL223 and S e r 264
(Oettmeier 1992) and IleL229 and Leu 271 (Ohad and Hirschberg 1992), respectively. In this study electron transfer, quinone binding and inhibition measurements were carried out on spontaneous atrazin resistant mutant of Rhodobacter sphaeroides R-26 (R/89). The mutation was located in the L subunit near to the binding site for QB: IleL229 Met. This amino acid is not involved directly in the proton transfer to QB, but makes the QB binding stable being in van der Waals contact with it. The R/89 mutant is the carotenoidless equivalent of the mutant isolated earlier from Rhodobacter sphaeroides 2.4.1. by Paddock et al. 1988. Replacement of IleL229in other strains like Rb. capsulatus (Bylina and Youvan 1987; Baciou et al. 1993) and Rps. viridis (Sinning and Michel 1987) attracted special interest. The IleL229 ~ Ser mutation in Rb. capsulatus decreased the free energy of stabilization of the secondary semiquinone and lowered the binding affinity of UQ6 but did not cause resistance to inhibitors (Baciou et al. 1993). We determined the dissociation constants of water soluble quinone UQo, terbutryne and o-phenanthroline for the mutant R/89. The overlapping effects of altered electron transfer and thermodynamic properties of QB were separated from the changes in quinone binding affinity. It was observed that subtle structural changes in the mutant increased dramatically the dissociation constant of the quinone and, as a consequence, decreased the free energy of stabilization of the secondary semiquinone, QB-. The known structure of the native RC is used to deduce possible structural alterations in the mutant.
Materials and methods
Chemicals The buffers succinate, citrate, MES (2-[Nmorpholino]ethanesulfonic acid), MOPS (3-[Nmorpholino]propansulfonic acid), TRIS (2-amino-2(hydroxymethyl)-l,3-propanediol), BTP (1,3-bis(tris(hydroxymethyl)methylamino)-propane), CHES (2[N-cyclohexylamino] ethanesulfonic acid), CAPS (3(cyclohexylamino)-1-Propane-sulfonic acid) and horse heart ferricytochrome C were obtained from Sigma. Solutions of ferrocene and UQo (Sigma) were prepared in ethanol prior to use. Ubiquinone-50 (UQ10, 2,3-dimethoxy-5-methyl-6-decaisoprenyl-1,4benzoquinone, Sigma) was solubilized by sonication in
137 30% Triton X-100 (octylphenol-polyethyleneglycolether, Serva).
Strains and growth conditions Rhodobacter sphaeroides R-26 (R-26) strain was grown photoheterotrophically under anaerobic conditions in a medium supplemented with potassium succinate as described earlier (Ormerod et al. 1961). Spontaneous atrazin resistant R/89 strain was selected by transferring the wild type R-26 cells to the culture medium containing 200 #M atrazin. Cultures were then plated on to atrazin containing agar plates and incubated anaerobically in low light. Colonies developed on plates were used as inocula for photoheterotrophic growth in liquid medium. Growth temperature was 30 °C and the light intensity of ~ 90 #E m -2 s -1 was provided by tungsten lamps (Nagy et al. 1991). Cloning and sequencing the R/89 DNA p B S + / - DNA used for cloning and sequencing was prepared by an alkali lysis procedure (Maniatis et al. 1982). E. coli XL1-Blue (Bullock et al. 1987) was used as a host both for plasmid and phage vectors. Restriction endonucleases were obtained from New England BioLabs, proteinase K from Sigma and lysozyme from Reanal. ['7-32P]ATP was from Izinta, [a-35S] dATP and multiprime DNA labelling kit from Amersham. The hybridization probe was an ,-~ 850 bp XbaI-SalI fragment of a pUC 18 clone containing the R-26 pufL sequence except for the last 66 bases of the gene (a gift from Dr Mike Jones, University of Sheffield). An internal oligonucleotide primer with a sequence of Rb. sphaeroides 2.4.1.245L-9-51L non-coding strand (Williams et al. 1984) was synthesized in a Cyclone Plus (Milligene Biosearch) system. T7 DNA sequencing kit from Pharmacia and Sequenase version 2 from USB were used to determine the DNA sequence, and Microgenie (Beckmann) program was used for the analysis. Photoheterotrophically grown Rb. sphaeroides R/89 was harvested in early stationary phase. The genomic DNA was prepared following the method of Fornari and Kaplan (1983) and digested with PstI. XL1-Blue competent cells for cloning the 4-6 kb fraction were freshly prepared and transformed by the method of Chung et al. (1989). XL1-Blue frozen competent stock prepared by the same method was used for all the further transformation. Single standard DNA from pBS subclones was prepared by superinfection
by VCS M13 helper phage, and all the (single strand) templates were purified. The fragments resulted in the sequencing reactions were separated on 0.025 × 30 x 40 cm 5 or 6% denaturing polyacrylamide gels.
Preparation of chromatophores and reaction centers Cells were broken by French press ( ~ 150 MPa) and after removing unbroken cells and cell debris by centrifugation (20 min, ~ 40 000 × g) the chromatophores were sedimented by ultracentrifugation (90 min, 240 000 × g). Reaction centers were prepared by LDAO (lauryldimethylamine N-oxide, Fluka) solubilization and standard protein purification methods (ammonium sulphate precipitation, DEAE Sephacell (Sigma) column chromatography and ultrafiltration) as described previously (Mar6ti and Wraight 1988). RCs with one quinone were prepared as described by Okamura et al. (1975) with minor modifications. QB was reconstituted by adding a 10-fold excess of UQ10 to the RC solutions.
Optical measurements Flash-induced absorption changes were measured by a single-beam spectrophotometer of local design. The excitation was achieved by a xenon flash lamp (type EG&G FX200, tl/2 ~ 8.5 #s) passed through a 760 nm cut-off filter. The photomultiplier (EM19558) was protected by a Corning 4.96 blue filter. The analog signal of the photomultiplier was converted with an A/D converter card (Advantech PCL818) attached to an IBM-compatible 386 AT computer. The reconstitution of the secondary quinone activity of the RC was determined by decomposing the kinetic trace of the flash-induced absorption change due to the dark re-reduction of the oxidized primary donor P+ measured at 430 nm (Kleinfeld et al. 1984). The two-electron gate function of the acceptor quinone complex was investigated by two assays: semiquinone absorbance change measured at 450 nm (Vermeglio and Clayton 1977; Tandori et al. 1991) and cyt c 2+ oxidation detected at 550 nm (Kleinfeld et al. 1985) after trains of saturating flashes.
138 Results
46'
Cloning and sequencing The pulL containing PstI fragment appeared to be 4.5 kb by Southern hybridization, similarly to that found for Rb. sphaeroides 2.4.1. earlier (Williams et al. 1984). The PstI digested genomic DNA was separated on a 0.8% preparative agarose gel, and the electroeluated ,,~ 4-5 kb fraction was ligated into pBS+ linearized by PstI previously. Two of the screened 200 recombinants - resulted in the transformation of E. coli XL1-Blue with the ligation mixture above - were found to be positive by colony hybridization. However, only one of them contained the 4.5 kb PstI insert and gave positive signal, when purified DNA was analyzed by separation and hybridization in the agarose gel (Tsao et al. 1983). The 4.5 kb PstI insert was cut with PvuII, and both the 1.2 and 3.3 kb fragments were ligated into SmaI and PstI sites of pBS+. An ,-~ 1.7 kb SmaI PstI fragment of the 4.5 kb insert was subcloned similarly, and the entire sequence of the pufL was determined from the subclones. Comparing with the putL sequence published for the Rb. sphaeroides 2.4.1. the only difference was a C ~ G exchange on the noncoding strand resulted an IleL229 ---+Met point mutation. Since the same amino acid change has been published for Rb. sphaeroides 2.4.1. (Paddock et al. 1988), the R/89 seems to be its carotenoidless pair.
30' ~ 20' a 10' o
.
A
o.~
e-. c--
25
oe-
20
0
0:4
0'.s
1:2
116
2.0
B
ffl
-Q
