Electron Transfer in Cyanobacterial Photosystem I
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m302965200
ISSN1083-351X
AutoresWu Xu, Parag R. Chitnis, А. И. Валиева, Art van der Est, Klaus Brettel, Mariana Guergova-Kuras, Yulia Pushkar, Stephan G. Zech, D. Stehlik, Gaozhong Shen, Boris Zybailov, John H. Golbeck,
Tópico(s)Spectroscopy and Quantum Chemical Studies
ResumoThe directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of τ ≈ 300 ns while optical data resolve an additional minor kinetic component of τ ≈ 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is ∼3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1–, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX– in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch. The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of τ ≈ 300 ns while optical data resolve an additional minor kinetic component of τ ≈ 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is ∼3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1–, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX– in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch. The pathway of light-induced electron transfer among symmetrically placed electron transfer cofactors has been a long standing issue in the study of photosynthetic reaction centers (RCs). 1The abbreviations used are: RC, reaction center; PS, photosystem; EPR, electron paramagnetic resonance; Chl, chlorophyll; phylloquinone, 2-methyl-3-phytyl-1,4-naphthoquinone or 2-methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-1,4-naphthalenedione. Although RCs show considerable diversity between various organisms, all consist of a protein dimer with two branches of cofactors that extend across the membrane from a "special pair" of chlorophyll molecules on the donor side to a pair of quinones on the acceptor side. The whole arrangement has pseudo-C2 symmetry, however, differences in the amino acid sequences of the two proteins and/or positions and structures of the cofactors break the symmetry significantly in all heterodimeric RCs. In Type II RCs, quinones function as the terminal electron acceptors, and electron transfer is known to be highly asymmetric and strongly biased toward one branch (referred to as the A-branch). One apparent reason for this asymmetry is the fact that the quinone acceptor in the B-branch, QB, functions as a mobile electron carrier, and electron transfer down the A-branch stabilizes the electron while the QB site undergoes a structural reorganization needed to accept an electron (1Graige M.S. Feher G. Okamura M.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11679-11684Crossref PubMed Scopus (205) Google Scholar). In Type I RCs, there are no known mobile quinones, and both branches converge at the iron-sulfur cluster FX on the stromal side of the membrane. The electron is further transferred to the FA and FB iron-sulfur clusters, which function as the terminal electron acceptors. Because of these differences, the directionality of electron transfer in Type I RCs cannot be inferred from knowledge of Type II RCs. Unlike in the bacterial RC, the two branches in Photosystem I (PS I) are difficult, if not impossible, to distinguish spectroscopically. As discussed in the previous paper (2Xu W. Chitnis P. Vaieva A. van der Est A. Pushkar J. Teutloff C. Krzystyniak M. Zech S. Bittl R. Stehlik D. Zybailov B. Shen G. Golbeck J. J. Biol. Chem. 2003; 278: 27864-27875Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), the use of point mutants and time-resolved EPR and optical spectroscopy allows this issue to be addressed in PS I. The initial electron transfer steps are difficult to observe because the trapping of the excitation from the antenna masks these early events. In contrast, the subsequent electron transfer from phylloquinone (A1) to the iron-sulfur center FX is easily observed and provides a convenient way to study the kinetics and pathway of electron transfer. Early time-resolved optical studies of electron transfer from A1– to FX appeared to be contradictory, with kinetics from UV absorbance changes attributed to A1– reoxidation reported to have a t ½ of 15 ns in PS I particles isolated from spinach (3Mathis P. Sétif P. FEBS Lett. 1988; 237: 65-68Crossref Scopus (36) Google Scholar) and 200 ns in PS I particles isolated from Synechococcus sp (4Brettel K. FEBS Lett. 1988; 239: 93-98Crossref Scopus (87) Google Scholar), whereas transient EPR data on PS I particles from Synechococcus sp. and spinach chloroplasts both gave a value of τ = 260 ns (t ½ = 180 ns) (5Bock C.H. van der Est A.J. Brettel K. Stehlik D. FEBS Lett. 1989; 247: 91-96Crossref Scopus (57) Google Scholar, 6van der Est A. Bock C. Golbeck J. Brettel K. Sétif P. Stehlik D. Biochemistry. 1994; 33: 11789-11797Crossref PubMed Scopus (75) Google Scholar). Later studies using so-called PS I-β particles from spinach showed a biphasic decay attributed to A1– oxidation with t ½ of 25 and 250 ns, and relative amplitudes of 65 and 35%, respectively (7Sétif P. Brettel K. Biochemistry. 1993; 32: 7846-7854Crossref PubMed Scopus (88) Google Scholar). Both kinetic phases were attributed to electron transfer from A1– to FX. Studies at faster time scales with PS I particles from Synechocystis sp. PCC 6803 also showed an additional kinetic phase attributed to A1– oxidation with a t ½ of 10 ns (8Brettel K. Garag G. Photosynthesis: Mechanisms and Effects. Vol. I. Kluwer Academic Publishers, Budapest, Hungary1998: 611-614Google Scholar). Although the faster of the two kinetic phases cannot be resolved directly by transient EPR, changes in its relative amplitude are reflected in the observed spin polarization (9Kandrashkin Y.E. van der Est A. RIKEN Rev. 2002; 44: 124-127Google Scholar, 10van der Est A. Biochim. Biophys. Acta. 2001; 1507: 212-225Crossref PubMed Scopus (56) Google Scholar), which is sensitive to the spin dynamics of short-lived precursor states with lifetimes as short as 500 ps. Recently, this influence of the fast kinetic phase on the spin polarization patterns was investigated (9Kandrashkin Y.E. van der Est A. RIKEN Rev. 2002; 44: 124-127Google Scholar), and it was concluded that the amplitude of the fast phase in whole cells and PS I particles of cyanobacteria could account for at most ∼20% of the total amplitude, whereas the transient EPR spectra of PS I particles isolated from spinach and Chlamydomonas rheinhardtii show a much larger influence (9Kandrashkin Y.E. van der Est A. RIKEN Rev. 2002; 44: 124-127Google Scholar). Moreover, the ratio of the fast to slow kinetic phases is not constant between different preparations; rather, PS I particles isolated from spinach show a diminished amplitude of the optically detected 25-ns kinetic phase when isolated using less harsh conditions, down to 30% in a particle prepared without detergent (7Sétif P. Brettel K. Biochemistry. 1993; 32: 7846-7854Crossref PubMed Scopus (88) Google Scholar), whereas transient EPR suggests at most a very small contribution from the fast kinetic phase in spinach chloroplasts and cyanobacterial whole cells (6van der Est A. Bock C. Golbeck J. Brettel K. Sétif P. Stehlik D. Biochemistry. 1994; 33: 11789-11797Crossref PubMed Scopus (75) Google Scholar, 9Kandrashkin Y.E. van der Est A. RIKEN Rev. 2002; 44: 124-127Google Scholar, 10van der Est A. Biochim. Biophys. Acta. 2001; 1507: 212-225Crossref PubMed Scopus (56) Google Scholar). In contrast, recent optical studies of whole cells of C. reinhardtii (11Joliot P. Joliot A. Biochemistry. 1999; 38: 11130-11136Crossref PubMed Scopus (163) Google Scholar) showed biphasic kinetics attributed to the reoxidation of A1– with t ½ of 18 ns and t ½ of 160 ns and of nearly equal amplitude. Hence, it appears that the fast kinetic phase is a property inherent to PS I but the ratio of the amplitudes of the fast and slow kinetic phases is species-dependent and sensitive to environmental conditions such as the presence of the detergent. Moreover, the sensitivity to detergent isolation is much higher in eukaryotic PS I compared with cyanobacterial PS I. Whereas the biphasic kinetics observed in the near UV are now generally thought to result from electron transfer from A1– to FX, the origin of the biphasic behavior remains controversial (see Ref. 12Brettel K. Leibl W. Biochim. Biophys. Acta. 2001; 1507: 100-114Crossref PubMed Scopus (294) Google Scholar for review). Sétif and Brettel (7Sétif P. Brettel K. Biochemistry. 1993; 32: 7846-7854Crossref PubMed Scopus (88) Google Scholar) suggested that the redox potentials of A1 and FX are close and that the fast kinetic phase reflects the establishment of a redox equilibrium between A1 and FX. This proposal was made prior to detailed knowledge about the pseudo C2 symmetry of PS I, and therefore presupposes a unidirectional pathway of electron transfer. Joliot and Joliot (11Joliot P. Joliot A. Biochemistry. 1999; 38: 11130-11136Crossref PubMed Scopus (163) Google Scholar) suggested that the biphasic kinetics could come about from either two conformational states that differ by the reoxidation rate of A1– or two phylloquinones that correspond to the two branches of the PS I heterodimer involved in electron transfer. The former presupposes unidirectional electron transfer and the latter presupposes bidirectional electron transfer. The latter idea arose when the model of PS I based on the 6-Å crystal structure (13Krauß N. Hinrichs W. Witt I. Fromme P. Pritzkow W. Dauter Z. Betzel C. Wilson K.S. Witt H.T. Saenger W. Nature. 1993; 361: 326-331Crossref Scopus (305) Google Scholar) showed the presence of a 2-fold axis of symmetry similar to the pseudo 2-fold axis of symmetry in the purple bacterial RC. The atomic resolution structure based on the 2.5-Å electron density map (14Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauß N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2081) Google Scholar, 15Fromme P. Jordan P. Krauss N. Biochim. Biophys. Acta. 2001; 1507: 5-31Crossref PubMed Scopus (378) Google Scholar) shows that the position of the electron transfer cofactors and the identity and positions of nearby amino acids are highly similar on the PsaA side and the PsaB side polypeptides. In particular, the phylloquinones on the PsaA side and the PsaB side are located in similar environments that consist of: (i) a H-bond between a backbone Leu and the stromal-facing carbonyl group of phylloquinone in the ortho position relative to the phytyl tail, (ii) a π–π interaction with a Trp, and (iii) an apparent lack of a H-bond to the other phylloquinone carbonyl group in the meta position relative to the phytyl tail. One significant break in symmetry is in the P700 chlorophyll a′/a special pair; the chlorophyll a′ has three H-bonds to PsaA side amino acids, whereas there are no H-bonds to the chlorophyll a on the PsaB side. Other significant symmetry breaks are also found further down the electron transfer chain in the high resolution structure (15Fromme P. Jordan P. Krauss N. Biochim. Biophys. Acta. 2001; 1507: 5-31Crossref PubMed Scopus (378) Google Scholar). The extent to which the two quinones are active in the electron transport chain, the origin of the biphasic kinetics of A1– reoxidation, and the role that the structural features of the binding site play in determining the electron transfer kinetics are all unresolved issues. In this paper, we report EPR and optical kinetic studies of mutants in and around the quinone binding sites of PS I. The premise of these experiments is that a change in the environment of the quinone should lead to a change in its redox potential, which, in turn, should translate to a change in the forward kinetics of electron transfer from the quinone to the iron-sulfur clusters. A network of H-bonded residues extends from the Met axial ligand of A0 through A1 to FX. The residues involved in this network are thus candidates for point mutations, which are expected to influence the rate of forward electron transfer. In the preceding paper (2Xu W. Chitnis P. Vaieva A. van der Est A. Pushkar J. Teutloff C. Krzystyniak M. Zech S. Bittl R. Stehlik D. Zybailov B. Shen G. Golbeck J. J. Biol. Chem. 2003; 278: 27864-27875Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), we showed that the point mutations W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB cause only subtle structural and electronic changes so that they act as suitable markers for following the pathway of electron transfer. As discussed in the preceding paper (2Xu W. Chitnis P. Vaieva A. van der Est A. Pushkar J. Teutloff C. Krzystyniak M. Zech S. Bittl R. Stehlik D. Zybailov B. Shen G. Golbeck J. J. Biol. Chem. 2003; 278: 27864-27875Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), the W697FPsaA, W677FPsaB, S692CPsaA, and S672CPsaB, mutants affect the quinone binding sites and we expect only electron transfer in the branch containing the mutation to be affected. The R694APsaA and R674APsaB mutants on the other hand may affect both branches because each is involved in a salt bridge from the jk-surface loop to Gly572PsaB (Gly585PsaA) in the respective FX binding loop (16Antonkine M. Jordan P. Fromme P. Krauß N. Golbeck J. Stehlik D. J. Mol. Biol. 2003; 327: 671-697Crossref PubMed Scopus (47) Google Scholar). In other words, this residue ties together the region of the quinone with that of the iron-sulfur cluster. Mutations of these residues should disrupt this arrangement and would be expected to cause a change in the properties of FX. The results presented in this paper address the issue of directionality and biphasic kinetics and can be interpreted to show that in Synechocystis sp. PCC 6803, the majority of electrons proceed along PsaA side cofactors in PS I. Whereas we cannot rule out the participation of PsaB side cofactors in PS I electron transfer, we place an upper limit on the fraction of electrons taking this pathway. Mutants and Photosystem I Complexes—The mutant strains containing the W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB substitutions have been described previously (2Xu W. Chitnis P. Vaieva A. van der Est A. Pushkar J. Teutloff C. Krzystyniak M. Zech S. Bittl R. Stehlik D. Zybailov B. Shen G. Golbeck J. J. Biol. Chem. 2003; 278: 27864-27875Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Isolation of membranes and purification of PS I complexes was performed according to previously published procedures (2Xu W. Chitnis P. Vaieva A. van der Est A. Pushkar J. Teutloff C. Krzystyniak M. Zech S. Bittl R. Stehlik D. Zybailov B. Shen G. Golbeck J. J. Biol. Chem. 2003; 278: 27864-27875Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Flash-induced Transient Absorption Spectroscopy in the Near UV/Blue Region—Flash-induced absorbance changes of isolated PS I complexes were measured with a time resolution of about 2 ns with a set-up described previously (17Brettel K. Leibl W. Liebl U. Biochim. Biophys. Acta. 1998; 1363: 175-181Crossref PubMed Scopus (43) Google Scholar) using 300-ps pulses of about 300 nJ/cm2 at 532 nm for excitation (repetition rate, 1 Hz) and the relatively flat top of a 50-μs Xe flash as measuring light. Stock solutions of PS I complexes were diluted in a buffer containing 50 mm Tris, pH 8.3, 10 mm sodium ascorbate, and 500 μm 2,6-dichloroindophenol, to a final Chl concentration of typically 60 and 150 μm for measurements at 380 and 480 nm, respectively. The optical path length for the measuring light was 2 mm. Between 1024 and 4096 transients were averaged for each sample and wavelength to improve the signal-to-noise ratio. A Marquardt least squares algorithm program was used for fitting of the absorbance change transients to a multiexponential decay. Time zero was defined as the midpoint of the rising edge of the transient, and fitting was started 2.5 ns after time zero. Flash-induced absorbance changes in whole cells were measured with an optical setup described previously (18Béal D. Rappaport F. Joliot P. Rev. Sci. Instrum. 1999; 70: 202-207Crossref Scopus (82) Google Scholar). Cells were centrifuged and resuspended in 20 mm Tris at pH 8.2 in the presence of 5% Ficoll. Dichlorodimethylurea (20 μm) and 2 mm hydroxylamine were added to inactivate Photosystem II and 20 μm carbonyl cyanide p-trifluoromethoxyphenylhydrazone was added to prevent accumulation of transmembrane potential. X-band Transient EPR Spectroscopy at Room Temperature—Transient EPR experiments at room temperature were carried out using a modified Bruker ESP 200 spectrometer equipped with a home-built, broadband amplifier (bandwidth >500 MHz) for direct detection experiments. Light excitation was provided by a Continuum YAG/OPO laser system operating at 680 or 532 nm and 10 Hz. The EPR signals were digitized using a LeCroy LT322 500 MHz digital oscilloscope and transferred to a PC for storage and analysis. The samples were measured using a flat cell and a Bruker rectangular resonator and contained 1 mm sodium ascorbate and 50 μm phenazine methosulfate as external redox mediators. The response time of the system is governed by the band-width of the resonator and is estimated to be ∼50 ns; the decay of the spin polarization limits the accessible time range to times shorter than a few microseconds. Complete time/field data sets were collected and analyzed to determine the lifetimes of the species and their decay-associated spectra. To ensure that the isolation procedure does not influence the kinetics or polarization patterns, transient EPR measurements of whole cells and isolated particles from wild-type Synechocystis sp. PCC 6803 were compared. The observed spin polarization patterns (not shown) and kinetic traces are identical demonstrating that the isolation of the particles has no effect on the kinetics. X-band Transient EPR Spectroscopy at 260 K—X-band transient EPR experiments on frozen solutions at 260 K were carried out using a Bruker ER046 XK-T microwave bridge equipped with a Flexline dielectric resonator (6van der Est A. Bock C. Golbeck J. Brettel K. Sétif P. Stehlik D. Biochemistry. 1994; 33: 11789-11797Crossref PubMed Scopus (75) Google Scholar) and an Oxford liquid helium gas-flow cryostat. The loaded Q-value for this dielectric ring resonator was about Q = 3000, equivalent to a rise time of τr = Q/(2π × νmw) ≈ 50 ns. The samples were illuminated using a Spectra Physics Nd-YAG/MOPO laser system operating at 10 Hz and contained 1 mm sodium ascorbate and 50 μm phenazine methosulfate. Our main effort focuses on applying a set of complementary spectroscopic techniques to characterize transient charge separated states in a comparative study of the influence of mutations near the A1 binding site. We begin with studies of optical absorbance changes in the near UV/blue in both whole cells and in PS I complexes and continue with transient EPR spectroscopy. Time-resolved Optical Measurements in Whole Cells and Isolated PS I Complexes—The advantage of studying kinetic processes in whole cells is that there is no potential damage to the acceptor cofactors as a result of detergent solubilization of the thylakoid membranes. The disadvantage is that the cells are actively growing and dividing, and PS I complexes may be present in a number of developmental states, including those undergoing assembly and those undergoing degradation. To minimize any potential for heterogeneity because of turnover and to reduce effects of background absorption and competing photoprocesses in larger antenna systems, studies were performed on whole cells and purified on PS I complexes. Ideally, the kinetic results should match in both studies. The flash-induced difference spectrum of A1–/A1 shows a broad absorption increase between 340 and 400 nm (4Brettel K. FEBS Lett. 1988; 239: 93-98Crossref Scopus (87) Google Scholar, 19Brettel K. Sétif P. Mathis P. FEBS Lett. 1986; 203: 220-224Crossref Scopus (92) Google Scholar). Measurements on PS I complexes were made at 380 and 480 nm, an absorption band in the visible spectrum that reflects the reduction of A1. For technical reasons, measurements were performed at 390 and 400 nm on whole cells. Fig. 1 depicts the decay of the relative flash-induced absorbance change at 400 nm on a logarithmic time scale in whole cells of the wild-type in comparison with the W697FPsaA (top) and W677FPsaB (bottom) mutants. Table I summarizes the lifetimes and relative amplitudes as obtained from a fit of the data to the sum of a fast and slow kinetic phase. At detection wavelengths of 390 and 400 nm, two kinetic phases are found in the wild type with similar time constants of τ = 10 and τ = 300 ns in an amplitude ratio of about 2:3. In the PsaA side mutant W697FPsaA the lifetime of the slow kinetic phase is increased by a factor of about 4 but the lifetime of the fast kinetic phase is relatively unchanged. In the corresponding (symmetric) PsaB side mutant W677FPsaB the lifetime of the slow kinetic phase is not changed significantly at either wavelength (see Table I) but the lifetime of the fast kinetic phase is increased by a factor of about 2.7 (see Fig. 1, top).Table IKinetic analysis of flash-induced absorbance changes attributed to A1- reoxidation in whole cells390 nm400 nmτRelative amplitudeτRelative amplitudeτRelative amplitudeτRelative amplitudensnsnsnsWT100.403000.60110.343400.66W697FPsaA110.4212800.58120.2712000.73W677FPsaB270.364000.64290.383800.62S692CPsaAaIn this mutant, a fast kinetic component (τ = 30 ns) was observed at 430 nm. The fast decrease of the signal may suggest that some PS I complexes undergo a charge recombination from P700+A0-.150.3513400.65140.3811400.62S672CPsaB130.382100.62150.412400.59R674APsaB110.3013000.70120.3213000.68a In this mutant, a fast kinetic component (τ = 30 ns) was observed at 430 nm. The fast decrease of the signal may suggest that some PS I complexes undergo a charge recombination from P700+A0-. Open table in a new tab Examples of flash-induced absorption changes at 380 nm measured in isolated PS I complexes with a time resolution of about 2 ns are shown in Fig. 2. All samples studied showed an instrument limited rise of absorption (including the formation of A1– (4Brettel K. FEBS Lett. 1988; 239: 93-98Crossref Scopus (87) Google Scholar, 19Brettel K. Sétif P. Mathis P. FEBS Lett. 1986; 203: 220-224Crossref Scopus (92) Google Scholar)). As in whole cells, the subsequent decay of the signal (attributed to electron transfer from A1– to the ironsulfur clusters (3Mathis P. Sétif P. FEBS Lett. 1988; 237: 65-68Crossref Scopus (36) Google Scholar, 4Brettel K. FEBS Lett. 1988; 239: 93-98Crossref Scopus (87) Google Scholar, 7Sétif P. Brettel K. Biochemistry. 1993; 32: 7846-7854Crossref PubMed Scopus (88) Google Scholar, 8Brettel K. Garag G. Photosynthesis: Mechanisms and Effects. Vol. I. Kluwer Academic Publishers, Budapest, Hungary1998: 611-614Google Scholar, 11Joliot P. Joliot A. Biochemistry. 1999; 38: 11130-11136Crossref PubMed Scopus (163) Google Scholar)) was affected by the mutations. The wild-type signal (Fig. 2) could be well fitted with two exponential decay phases of τ = 10.6 and τ = 240 ns at an amplitude ratio of about 1:3 (see Table II), in line with a previous report (8Brettel K. Garag G. Photosynthesis: Mechanisms and Effects. Vol. I. Kluwer Academic Publishers, Budapest, Hungary1998: 611-614Google Scholar). 2A slight bleaching present at the end of the depicted time scale is presumably from the state P700+(FA/FB)– and/or a small number of antenna chlorophyll triplets. Compared with the measurements on whole cells from the wild type at 390 and 400 nm (see Table I), the lifetimes of the two phases are rather similar, but the relative amplitude of the faster phase appears to be smaller in isolated PS I (but only at 380 nm, not at 480 nm). For the mutants W697FPsaA and W677FPsaB, the kinetics in isolated PS I complexes followed the same trends as in whole cells, i.e. the slower phase alone was slowed in the PsaA side mutant, whereas the faster phase alone was slowed in the PsaB side mutant. The relative amplitudes of the two phases were not significantly affected by these mutations (data not shown; see Table II for fit results). Similar results were observed in mutants of C. reinhardtii (20Guergova-Kuras M. Boudreaux B. Joliot A. Joliot P. Redding K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4437-4442Crossref PubMed Scopus (271) Google Scholar), in which the mutation of Trp to Phe on PsaA increased the lifetime of the slow phase while the corresponding (symmetric) mutation on PsaB slowed down the fast phase.Table IIKinetic analysis of flash-induced absorbance changes attributed to A1- reoxidation in isolated PS I complexes380 nm480 nmτRelative amplitudeτRelative amplitudeτRelative amplitudeτRelative amplitudensnsnsnsWT110.252400.759.80.392300.61W697FPsaA8.70.267200.748.10.388800.62W677FPsaB320.242600.76230.412600.59S692CPsaAaA significant evolution of the kinetics during prolonged repetitive excitation was observed for this mutant. The data presented in the table result from accumulations of 2048 transients (380 nm) and 1024 transients (480 nm) measured with freshly prepared samples. For a sample that had received about 6000 excitation flashes, a biexponential fit of the 380-nm signal yielded lifetimes (relative amplitudes) of 22 (0.58) and 580 ns (0.42). A significantly better fit was obtained with three exponentials: 9.4 (0.38), 61 (0.30), and 830 ns (0.32). The error in τ is estimated to be 20%.120.2910400.717.70.3813300.62S672CPsaB250.262800.74170.432500.57a A significant evolution of the kinetics during prolonged repetitive excitation was observed for this mutant. The data presented in the table result from accumulations of 2048 transients (380 nm) and 1024 transients (480 nm) measured with freshly prepared samples. For a sample that had received about 6000 excitation flashes, a biexponential fit of the 380-nm signal yielded lifetimes (relative amplitudes) of 22 (0.58) and 580 ns (0.42). A significantly better fit was obtained with three exponentials: 9.4 (0.38), 61 (0.30), and 830 ns (0.32). The error in τ is estimated to be 20%. Open table in a new tab The lifetimes and amplitudes as obtained from a fit of the data to a fast and slow kinetic phase in whole cells of the wild-type, S692CPsaA, and S672CPsaB mutants are summarized in Table I. In the PsaA side mutant S692CPsaA, the lifetime of the slow kinetic phase is increased by a factor of about 4, whereas within experimental error the lifetime of the fast kinetic phase does not appear to be lengthened. In the corresponding (symmetric) PsaB side mutant S672CPsaB, the life-time of the slow kinetic phase is not changed significantly at either wavelength (see Table I). Surprisingly, within experimental error the lifetime of the fast kinetic phase also does not appear to be lengthened at either detection wavelength. Note particularly that the large effect on the slow kinetic phase of the S692CPsaA mutant is not mirrored by that on the fast kinetic phase in
Referência(s)