- Email: [email protected]
Time-resolved spectroscopy of photosynthetic systems. 3: photosystem 1 preparations from the cyanobacterium Chlorogloea fritschii
Journal of Photochemistry
and Photobiology,
B: Biology, 5, (1990)
445 - 455
445
TIME-RESOLVED SPECTROSCOPY OF PHOTOSYNTHETIC SYSTEMS. 3: PHOTOSYSTEM 1 PREPARATIONS FROM THE CYANOBACTERIUM Chlorogloea fritschii RAYMOND SPARROW, ,ROBERT G. BROWfl
and E. HILARY
Schools of Applied Biology and Chemistry, Lancashire Polytechnic, Lancashire PRl 2TQ (U.K.)
EVANS Preston,
DAVID SHAW SERC Daresbury Laboratory,
Warrington WA4 4AD (U.K.)
(Received July 30, 1989; accepted October 12, 1989)
Keywords. Photosystem 1, time-resolved Chlorogloea fritschii, redox effects.
fluorescence,
cyanobacteria,
Summary Fluorescence decay profiles for a photosystem 1 preparation from the cyanobacterium Chlorogloea fritschii are reported. The decay profiles are dominated by a short-lived decay component of some 20 - 30 ps, but also exhibit two other components of lifetimes 400 - 650 ps and 3.0 - 3.6 ns. Time-resolved spectra of the three components of the decay are reported and are assigned principally to chlorophyll-protein complexes with various degrees of coupling to the reaction centre. The effect of redox potential on photosystem 1 is reported. It is concluded that there is a species in the energy transfer chain intermediate between the main light-harvesting pigments and P,,,a. “.
1. Introduction The fluorescence that is observed from photosynthetic organisms can be an extremely useful probe of the processes taking place within the system, but is of a complex nature both from the spectroscopic and kinetic viewpoint. Considerable progress has been made recently in the time scale encompassing the first few nanoseconds immediately following absorption of a photon, in that a number of fluorescent species have been shown to
TAuthor to whom correspondence should be addressed. loll-1344/90/$3.50
@ Elsevier Sequoia/Printed in The Netherlands
446
contribute to the fluorescence on the basis of the complex fluorescence decays that are observed (see, for example, refs. 1 - 4 and references cited therein). Analysis of the fluorescence decays of whole organisms or of higher plant thylakoid or chloroplast preparations has increased in complexity, such that three [ 1 - 41, four [2 - 41 or even five [ 51 exponential components may be required to explain alI the data fully. The majority of fluorescent components have been assigned to photosystem 2 (PS2) with a small contribution from photosystem 1 (PSl). One of the obvious ways to provide further information on these various species is to study the kinetic properties of the fluorescence of photosynthetic preparations enriched in PSl or PS2 and/or of mutants which are deficient in one or more of the components of the overall mechanism. With respect to PSl, Owens et al. [6] have reported decay times of 15 - 40 ps and 5 - 6 ns for PSl antenna chlorophylls with and without a trap respectively. The same group have previously shown that the shorter of these two decay times is linearly related to the size of the antenna chlorophyll pool [7]. The presence and lifetime of this component have been confirmed by Wittmershaus et al. [8] and Hodges and Moya [ 91. However, both of these groups have also observed a component with a decay time of some 100 ps which comprises 10% - 20% of the overall decay. .In addition, Hodges and Moya have also reported small contributions from two other longer-lived components, but none of these have lifetimes as long as the 5 - 6 ns reported by Owens et al. With the exception of Holzwarth [5], fluorescent components with lifetimes less than 100 ps have not been observed in intact algae or isolated chloroplasts [l - 41. We have previously reported [l] fluorescence decay results for lettuce chloroplasts and have attempted to simplify our experiments by subdividing the lettuce chloroplasts into PSl and PS2. In the latter case we have been successful and our results have been reported elsewhere [lo] ; however, to date, we have been unable to make a PSl preparation from lettuce that is satisfactory in terms of its stability with respect to fluorescence emission characteristics, even when the photochemical characteristics of PToOare unchanged. Therefore we have used a PSl preparation from the cyanobacterium Chlorogloea fritschii which has good stability and has been characterized previously [ll]. In a series of parallel experiments on this preparation over the same time scale, the changes in absorption induced by a 4 ps laser pulse have been studied. This technique provides complementary information to that obtained from the fluorescence measurements. In this paper, fluorescence studies on this preparation using synchrotron radiation excitation and single-photon counting detection are reported. 2. Experimental details Chlorogloea fritschii was maintained and grown and PSl was extracted as described previously [ 111. The resulting preparations had a chlorophyll a
447
ratio of approximately 5O:l. The concentrated preparation was to p700 either used immediately or stored at 193 K until required. Samples for fluorescence measurements were prepared by diluting the concentrated preparation with buffer (containing 0.06 M Tris and 0.03 M EDTA, pH 7.8) to give a chlorophyll concentration of approximately 10.0 pg cmd3 of chlorophyll a prior to measurement of the decay profile. Fluorescence decay profiles were measured as described previously [l] over the wavelength range 680 - 720 nm using interference filters at 10 nm intervals. The profiles were analysed in terms of a sum of exponential components as described previously [ 1, lo]. Time-resolved spectra were obtained by calculating the number of counts per second for each component of the decay at each wavelength and correcting for the transmission of the filters. Correction for the wavelength response of the photomultiplier was not attempted. Steady state fluorescence spectra were measured as described previously [ 121. 3. Results and discussion The fluorescence of three exponential
decay profiles of our PSl preparation require a sum components to achieve an acceptable fit between
X10’ I
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Fig. 1. Fluorescence decay profile of PSl excited at 440 nm with emission monitored at 680 nm fitted to a three exponential model (residual plot (a) and autocorrelation plot (b)). Residual plots for one and two exponential fits are also shown (plots (c) and (d) respectively).
449
calculation and experiment. A typical profile is shown in Fig. 1, and the decay parameters for the preparation as a function of wavelength are given in Table 1. The values in the table are the averaged results from a number of experiments; typically at least six separate measurements were taken and usually more than ten. The profiles were analysed both individually and globally using the SPLMOD package [13]. The lifetime values which are obtained from individual analyses of the decay curves are sufficiently similar to indicate that global analysis is warranted. However, the lifetime of the fastest component varies quite considerably from experiment to experiment. This variation is not reduced (in terms of its standard deviation) on global analysis, but more extreme values of the computed lifetime are eliminated. We ascribe this variability, which is not observed to the same extent in the other components, to the time-resolution limitations imposed on the experiments by the synchrotron and single-photon counting technique. Given the pulse duration of the synchrotron [l], lifetimes of the order of 20 ps must be viewed with caution, but the agreement between our value and those obtained by other workers is good enough to encourage confidence that the observed 20 ps lifetime is real. In our later measurements, the synchrotron source was sufficiently stable to allow us to collect decay data at all emission wavelengths with the excitation intensity constant. This enabled us to construct time-resolved emission spectra for the three components and these are shown in Fig. 2. All three time-resolved spectra exhibit a peak at 690 nm and the two shorter lifetime components also have a peak or shoulder at 710 nm. The positions of both of these spectral features are in agreement with the steady state spectrum, but the intensity of the longer wavelength emission appears to be rather greater than would be anticipated on the basis of the steady state spectrum. This spectral
TABLE 1 Fluorescence fritschiP
lifetimes
(ps) and percentage
Data sets analysed
h(nm)
71
Pl
Individually
680 690 700 710 720
15 fa 21+l3 23+6 26 f 5 26 f 3
64 75 80 83 85
Globally
680 690 700 710 720
22+10 22f10 22-+10 22 * 10 22-+10
55+10 71+6 81f7 8524 89f4
contributions
72
f f + f k
11 5 6 3 4
for PSI from Chlorogloea
p2
73
p3
605 515 575 660 410
+ * f * f
105 130 70 40 80
15*2 12+2 13f2 9+2 9 f 2
3500f240 3400-+380 2930f350 3610f430 3260 f 480
21 f 6 13 + 6 7k5 7+2 5f3
550 550 550 550 550
2 + + + f.
100 100 100 100 100
1723 lOf3 14f3 10+5 6 * 3
3360f400 3350+400 3350f400 3350-+400 3350 f 400
28 k 8 19 f 6 5*3 5f3 5+3
Vhe + values are the average standard deviations for the data sets analysed.
450
I
680
700 Wavelength
720 (mu)
Fig. 2. Time-resolved fluorescence ponents for PSl alone.
spectra of short (0), middle (A) and long (v) com-
feature is the subject of further investigation as we attempt to make substantial improvements in the wavelength resolution of the spectra. In addition to the fluorescence work on PSl that is summarized in Section 1, there have been a number of picosecond flash absorption studies of PSl and the kinetics of some of the component species assigned to the light-harvesting system and the reaction centre are known. Porter and coworkers [14, 151 and Il’Ina et al. [16] have reported three component kinetics for the deactivation of the antenna chlorophylls with lifetimes of 15 - 20 ps [14, 151 or 20 - 45 ps [16], approximately 300 ps and greater than 500 ps. Nuijs et al. [17] have reported a 40 + 5 ps lifetime for antenna chlorophyll decay, but this could be limited by the pulse width of their laser (full width at half maximum (FWHM), 35 ps). The excitation flash intensity used during these experiments is also variable, so any discrepancies might also be ascribed to the formation of different excited states in different proportions. We have previously reported complementary antenna decay and P,oo+ rise times of 20 ps [18], similar to the results of Porter and coworkers [14, 151. However, our most recent results [ 191 indicate that the
451
decay of the antenna pigments measured at 675 nm has a lifetime in the range 0.2 - 4.0 ps, which is difficult to resolve with our current 4 ps pulse width. This suggests that fluorescence transients ascribed to this energy transfer process may need further consideration, or that two or more lightharvesting to reaction centre energy transfer processes may be involved. Indeed, the spectral dependence of our fluorescence decay components (Fig. 2) suggests that this might be the case. All of the observed components can be assigned to fluorescence from antenna chlorophylls and complexes close to P,,. The longest-lived component of some 3.0 ns is believed to be a chlorophyll-protein complex which is completely uncoupled from the rest of the antenna and from the reaction centre. We do not believe that it is due to free chlorophyll solubilized in the detergent used in the extraction for two reasons: (i) the lifetime is a factor of two shorter than the 5.0 - 6.0 ns lifetime observed for chlorophyll a in the detergents that are used in the extraction procedure [20] and (ii) the emission is red shifted by some 10 - 20 nm from that of detergentsolubilized chlorophyll. Addition of sodium dodecyl sulphate to the PSl preparation results in a huge increase in fluorescence intensity and a decay profile which is virtually a single exponential with a lifetime of approximately 5.0 ns. Owens et al. [6,7] reached the same conclusion about the 6.0 ns component observed in PSl preparations from Chlamydomolzus reinhardtii. Our 3.0 ns lifetime is in good agreement with those assigned by Green et al. [21], Suter et al. [22] and Fleming and coworkers [23,24] to uncoupled chlorophyll. Presumably the decrease in lifetime from 6.0 to approximately 3.0 ns is due to a small amount of. residual energy transfer between the chlorophylls in the uncoupled protein complex or is an intrinsic property of chlorophyll a when complexed to a protein. In preparations from C. reinhardtii, Fleming’s group have observed three fluorescence components when the PSl preparation has a chlorophyll to P7c0 ratio of greater than 40 [6]. Concurrent with the appearance of this middle component of lifetime 250 - 2500 ps, the preparation also contains chlorophyll b. C. fritschii contains no chlorophyll b; on the basis of the similarity of the emission spectrum of the 400 - 650 ps component with that of the 3.0 ns component, we assign it to antenna complexes in which the coupling to the reaction centre has been partially disrupted. There seems little doubt that the short-lived fluorescence component is due to fluorescence from antenna complexes where energy transfer to the reaction centre is fully operational. However, based on the fast absorption changes observed at 675 nm [19], the 20 ps fluorescence cannot originate in the bulk antenna pigments. However, transient absorption changes are also observed in the wavelength region 670 - 720 nm with lifetimes of some 30 - 50 ps. These may well result from the antenna pigment complexes present in the model proposed by Wittmershaus [25] where the main antenna complexes absorbing at 680 nm (&so) and fluorescing at 690 nm pass their energy to a complex which is intermediate between them and P ,oO, This latter complex absorbs at 697 nm (Chg7) and fluoresces at 720 nm.
452
Our 20 ps component exhibits peaks at both 690 and 710 - 720 nm and we believe that this may be due to fluorescence from both C6s0 and Cbg7. The short-lived fluorescence component may be comprised of two separate decay components from these two species. Alternatively, the fluorescence from C6s0 may be derived mainly from back transfer of energy from C& and the fluorescence kinetics of C6s0 may therefore be determined by those of the C& precursor. If the lifetime values in Table 1 for the individual data analyses (rather than the global analysis) are examined, there is some evidence that they increase with wavelength. If, on the basis of the absorption data, we assign a lifetime of 0.2 - 4.0 ps to C6s0and a lifetime of 40 - 50 ps to C@T, the change in the fluorescence lifetime values as a function of wavelength could be explained by there being relatively more of the C&, fluorescence at longer wavelength. However, these very fast processes are really straining the limits of time resolution for the timecorrelated, single-photon counting technique, even with a fast laser as the excitation source. It is clear that a subpicosecond laser, together with sensitive streak camera detection, is required to probe these time scales properly. In an attempt to obtain further insight into the origin of the PSl fluorescence and the mechanisms that are operating, we added redox reagents to alter the oxidation state of P,as. In particular, we added potassium ferricyanide (6 mM) to oxidize P,aO and sodium dithionite to reduce P,oo and partially reduce the iron-sulphur acceptor complex [ 261. In both cases the lifetime of the short-lived component remains in the region of 20 ps, but the spectrum of this component varies considerably from that observed for PSl alone. The lifetime of the middle component increases to 1.2 ns but its spectrum is essentially unchanged. This is also true of the longer-lived component whose lifetime increases to 4.0 - 5.0 ns. In the case of the middle and long-lived components, it appears that the addition of the two redox chemicals further disrupts the pigment-protein complexes so that the fluorescence lifetimes increase but the spectra remain unchanged. The time-resolved fluorescence spectra of the short component of PSl with and without added potassium ferricyanide and sodium dithionite are compared in Fig. 3. The three spectra are drawn to enable comparison of the overall shapes of the spectra. The same vertical scale has not been used for each spectrum because of unquantifiable variations in excitation intensity between the three sets of data. The three spectra compared in Fig. 3 substantiate the suggestion made earlier that we are observing short-lived fluorescence from two species, both of which lie in the path of transfer of excitation energy to PToO.In the absence of any additions it is clear from Fig. 3 that the redox state of P,OOis intermediate between fully oxidized and fully reduced. When P,,,,, is reduced (in the presence of sodium dithionite), Cm0 is less fluorescent than Cbg7. This situation is reversed when PToOis oxidized. This is unexpected because if P,OOis oxidized, and therefore unable to accept
453 I
I
PSl + Dithionite
P 6130
700
720
1
I
PSl + Ferricyanide
PSl Alone
r 680 700 Wavelength
720 (nm)
680
700
Fig. 3. Time-resolved fluorescence spectra of the short-lived component presence and absence of potassium ferricyanide and sodium dithionite.
720
of PSl in the
excitation energy from C&,, the latter becomes the terminal chromophore in the energy transfer chain and should fluoresce much more strongly than when PYoo is reduced and able to accept energy. However, if C& is the terminal chromophore it will have a longer fluorescence lifetime than when it is coupled to P,,, and this may be the cause of the lengthening of the lifetime of the middle component in the presence of potassium ferricyanide. Alternatively, the ferricyanide may also oxidize C6s, and thereby cause it to be less fluorescent. It is clear that further work is needed to resolve completely the fluorescence properties of PSl. However, the evidence that we have to date supports our proposal of a species in the energy transfer chain which is intermediate between PToo and the bulk of the light-harvesting pigments [271. Acknowledgments We wish to thank the Science and Engineering Research Council and the Royal Society for financial support and Professors G. Fleming and I. Moya and Drs. B. Wittmershaus and A. Holzwarth for providing preprints of their work.
454
References
1 R. Sparrow, R. G. Brown, E. H. Evans and D. Shaw, Time-resolved fluorescence spectroscopy of photosynthetic systems using synchrotron radiation. Part 1. Fluorescence kinetics of lettuce chloroplasts, J. Chem. Sot., Faraday Trans. 2, 82 (1986) 2249 - 2267. 2 A. R. Holzwsrth, J. Wendler and W. Haehnel, Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of photosystem 1 fluorescence, Biochim. Biophys. Acta, 807 (1985) 155 - 167. 3 M. Hodges and I. Moya, Time-resolved chlorophyll fluorescence studies of photosynthetic membranes: resolution and characterisation of four kinetic components, Biochim. Biophys. Acta, 849 (1986) 193 - 202. 4 A. R. Holzwarth, Fluorescence lifetimes in photosynthetic systems, Photochem. Photobiol., 43 (1986) 707 - 725. 5 A. R. Holzwarth, personal communication. 6 T. G. Owens, S. P. Webb, R. S. Alberte, L. Mets and G. R. Fleming, Antenna structure and excitation dynamics in photosystem I. I. Studies of detergent-isolated photosystem I preparations using time-resolved fluorescence analysis, Biophys. J., 53 (1988) 733 - 745. 7 T. G. Owens, S. P. Webb, L. Mets, R. S. Alberte and G. R. Fleming, Antenna size dependence of fluorescence decay in the core antenna of photosystem I: estimates of charge separation and energy transfer rates, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 1532 - 1536. 8 B. P. Wittmershaus, D. S. Berns and C. Huang, Picosecond time-resolved fluorescence from detergent-free photosystem I particles, Biophys. J., 532 (1987) 829 - 836. 9 M. Hodges and I. Moya, Time-resolved chlorophyll fluorescence studies on pigmentprotein complexes from photosynthetic membranes, Biochim. Biophys. Acta, 935 (1988) 41- 52. 10 R. Sparrow, E. H. Evans, R. G. Brown and D. Shaw, Time-resolved spectroscopy of photosynthetic systems using synchrotron radiation. II. Photosystem 2 preparations from lettuce, J. Photochem. Photobiol. B, 3 (1989) 65 - 79. 11 C. A. Pullin and E. H. Evans, A requirement for EDTA in the separation of photosystems 1 and 2 from the cyanobacterium Chlorogloea fritschii, Biochem. J., 196 (1981) 489 - 493. 12 C. A. Pullin, R. G. Brown and E. H. Evans, Detection of allophycocyanin in photosystem I preparations from the blue-green alga, Chlorogloea fritschii, FEBS Lett., 101 (1979) 110 - 112. 13 R. W. Provencher and R. H. Vogel, Regularisation techniques for inverse problems in molecular biology, in P. Denflhard and E. Hairer (eds.), Progress in Scientific Computing, Vol. 1, Birkhauser, Boston, 1983, pp. 304 - 319. 14 L. B. Giorgi, T. Doust, B. L. Gore, D. R. Klug, G. Porter and J. Barber, Picosecond .absorption spectroscopy of photosystem I reaction centres from higher plants, Biochem. Sot. Trans., 14 (1986) 47 - 48. 15 B. L. Gore, T. A. M. Doust, L. B. Giorgi, D. R. Klug, J. P. Me, B. Crystal1 and G. Porter, The design of a picosecond flash spectroscope and its application to photosynthesis, J. Chem. Sot., Faraday Trans. 2,82 (1986) 2111 - 2115. 16 M. D. B’Ina, V. V. Krasauskas, R. J. Rotomskis and A. Yu Borisov, Difference picosecond spectroscopy of pigment-protein complexes of photosystem I from higher plants, Biochim. Biophys. Acta, 767 (1984) 501- 506. 17 A. M. Nuijs, H. J. van Gorkom, J. J. Plijter and L. N. M. Duysens, Primary charge separation and excitation of chlorophyll a in photosystem II particles from spinach as studied by picosecond absorption-difference spectroscopy, Biochim. Biophys. Acta, 848 (1986) 167 - 175.
455 18 E. H. Evans, R. Sparrow, R. G. Brown, D. Shaw, J. Barr, M. Smith and W. Toner, Fast fluorescence and absorption measurements of photosystem 1 from a cyanobacterium, Prog. Photosynth. Res., 1 (1987) 1.99 - 1.102. 19 R. W. Sparrow, E. H. Evans, R. G. Brown, M. C. W. Evans, W. Toner and R. Chittock, unpublished work. 20 R. G. Brown, E. H. Evans, S. G. Holderness, J. Manwaring and B. May, Fluorescence spectra and decay time measurements on chlorophyll a and a non-aggregating analogue, Photobiochem. Photobiophys., 5 (1983) 87 - 92. 21 B. R. Green, K. K. Karukstis and K. Sauer, Fluorescence decay kinetics of mutants of corn deficient in photosystem I and photosystem II, Biochim. Biophys. Acta, 767 (1984) 574 - 581. 22 G. W. Suter, P. Mazzola, J. Wendler and A. R. Holzwarth, Fluorescence decay kinetics in phycobilisomes from the blue-green alga Synechococcus 6301, Biochim. Biophys. Acta, 766 (1984) 269 - 276. 23 R. J. Gulotty, G. R. Fleming and R. S. Alberte, Low intensity picosecond fluorescence kinetics and excitation dynamics in barley chloroplasts, Biochim. Biophys. Acta, 682 (1982) 322 - 331. 24 R. J. Gulotty, L. Mets, R. S. Alberte and G. R. Fleming, Picosecond fluorescence study of photosynthetic mutants of Chlamydomonas reinhardtii: origin of the fluorescence decay kinetics of chloroplasts, Photochem. Photobiol., 41 (1985) 487 496. 25 B. P. Wittmershaus, Measurements and kinetic modelling of picosecond time-resolved fluorescence from photosystem I and chloroplasts, Prog. Photosynth. Res., 1 (1987) 1.75 - 1.82. 26 E. H. Evans, R. Cammack and M. C. W. Evans, Properties of the primary electron acceptor complex of photosystem I in the .blue green alga Chlbrogloea fritschii, Biochem. Biophys. Res. Commun., 68 (1976) 1212 - 1218. 27 E. H. Evans, R. Sparrow, R. G. Brown, R. Chittock, A. Langley, J. Barr, W. Toner and M. C. W. Evans, Picosecond absorption measurements of photosystem 1 from the cyanobacterium Chlorogloea fritschii, EBEC Reps., 5 (1988) 174.
and Photobiology,
B: Biology, 5, (1990)
445 - 455
445
TIME-RESOLVED SPECTROSCOPY OF PHOTOSYNTHETIC SYSTEMS. 3: PHOTOSYSTEM 1 PREPARATIONS FROM THE CYANOBACTERIUM Chlorogloea fritschii RAYMOND SPARROW, ,ROBERT G. BROWfl
and E. HILARY
Schools of Applied Biology and Chemistry, Lancashire Polytechnic, Lancashire PRl 2TQ (U.K.)
EVANS Preston,
DAVID SHAW SERC Daresbury Laboratory,
Warrington WA4 4AD (U.K.)
(Received July 30, 1989; accepted October 12, 1989)
Keywords. Photosystem 1, time-resolved Chlorogloea fritschii, redox effects.
fluorescence,
cyanobacteria,
Summary Fluorescence decay profiles for a photosystem 1 preparation from the cyanobacterium Chlorogloea fritschii are reported. The decay profiles are dominated by a short-lived decay component of some 20 - 30 ps, but also exhibit two other components of lifetimes 400 - 650 ps and 3.0 - 3.6 ns. Time-resolved spectra of the three components of the decay are reported and are assigned principally to chlorophyll-protein complexes with various degrees of coupling to the reaction centre. The effect of redox potential on photosystem 1 is reported. It is concluded that there is a species in the energy transfer chain intermediate between the main light-harvesting pigments and P,,,a. “.
1. Introduction The fluorescence that is observed from photosynthetic organisms can be an extremely useful probe of the processes taking place within the system, but is of a complex nature both from the spectroscopic and kinetic viewpoint. Considerable progress has been made recently in the time scale encompassing the first few nanoseconds immediately following absorption of a photon, in that a number of fluorescent species have been shown to
TAuthor to whom correspondence should be addressed. loll-1344/90/$3.50
@ Elsevier Sequoia/Printed in The Netherlands
446
contribute to the fluorescence on the basis of the complex fluorescence decays that are observed (see, for example, refs. 1 - 4 and references cited therein). Analysis of the fluorescence decays of whole organisms or of higher plant thylakoid or chloroplast preparations has increased in complexity, such that three [ 1 - 41, four [2 - 41 or even five [ 51 exponential components may be required to explain alI the data fully. The majority of fluorescent components have been assigned to photosystem 2 (PS2) with a small contribution from photosystem 1 (PSl). One of the obvious ways to provide further information on these various species is to study the kinetic properties of the fluorescence of photosynthetic preparations enriched in PSl or PS2 and/or of mutants which are deficient in one or more of the components of the overall mechanism. With respect to PSl, Owens et al. [6] have reported decay times of 15 - 40 ps and 5 - 6 ns for PSl antenna chlorophylls with and without a trap respectively. The same group have previously shown that the shorter of these two decay times is linearly related to the size of the antenna chlorophyll pool [7]. The presence and lifetime of this component have been confirmed by Wittmershaus et al. [8] and Hodges and Moya [ 91. However, both of these groups have also observed a component with a decay time of some 100 ps which comprises 10% - 20% of the overall decay. .In addition, Hodges and Moya have also reported small contributions from two other longer-lived components, but none of these have lifetimes as long as the 5 - 6 ns reported by Owens et al. With the exception of Holzwarth [5], fluorescent components with lifetimes less than 100 ps have not been observed in intact algae or isolated chloroplasts [l - 41. We have previously reported [l] fluorescence decay results for lettuce chloroplasts and have attempted to simplify our experiments by subdividing the lettuce chloroplasts into PSl and PS2. In the latter case we have been successful and our results have been reported elsewhere [lo] ; however, to date, we have been unable to make a PSl preparation from lettuce that is satisfactory in terms of its stability with respect to fluorescence emission characteristics, even when the photochemical characteristics of PToOare unchanged. Therefore we have used a PSl preparation from the cyanobacterium Chlorogloea fritschii which has good stability and has been characterized previously [ll]. In a series of parallel experiments on this preparation over the same time scale, the changes in absorption induced by a 4 ps laser pulse have been studied. This technique provides complementary information to that obtained from the fluorescence measurements. In this paper, fluorescence studies on this preparation using synchrotron radiation excitation and single-photon counting detection are reported. 2. Experimental details Chlorogloea fritschii was maintained and grown and PSl was extracted as described previously [ 111. The resulting preparations had a chlorophyll a
447
ratio of approximately 5O:l. The concentrated preparation was to p700 either used immediately or stored at 193 K until required. Samples for fluorescence measurements were prepared by diluting the concentrated preparation with buffer (containing 0.06 M Tris and 0.03 M EDTA, pH 7.8) to give a chlorophyll concentration of approximately 10.0 pg cmd3 of chlorophyll a prior to measurement of the decay profile. Fluorescence decay profiles were measured as described previously [l] over the wavelength range 680 - 720 nm using interference filters at 10 nm intervals. The profiles were analysed in terms of a sum of exponential components as described previously [ 1, lo]. Time-resolved spectra were obtained by calculating the number of counts per second for each component of the decay at each wavelength and correcting for the transmission of the filters. Correction for the wavelength response of the photomultiplier was not attempted. Steady state fluorescence spectra were measured as described previously [ 121. 3. Results and discussion The fluorescence of three exponential
decay profiles of our PSl preparation require a sum components to achieve an acceptable fit between
X10’ I
X10'
Fig. 1. (continued)
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-6 -8 100
200
300
400
500
CHANNEL
600
700
800
900
1000
NOS.
Fig. 1. Fluorescence decay profile of PSl excited at 440 nm with emission monitored at 680 nm fitted to a three exponential model (residual plot (a) and autocorrelation plot (b)). Residual plots for one and two exponential fits are also shown (plots (c) and (d) respectively).
449
calculation and experiment. A typical profile is shown in Fig. 1, and the decay parameters for the preparation as a function of wavelength are given in Table 1. The values in the table are the averaged results from a number of experiments; typically at least six separate measurements were taken and usually more than ten. The profiles were analysed both individually and globally using the SPLMOD package [13]. The lifetime values which are obtained from individual analyses of the decay curves are sufficiently similar to indicate that global analysis is warranted. However, the lifetime of the fastest component varies quite considerably from experiment to experiment. This variation is not reduced (in terms of its standard deviation) on global analysis, but more extreme values of the computed lifetime are eliminated. We ascribe this variability, which is not observed to the same extent in the other components, to the time-resolution limitations imposed on the experiments by the synchrotron and single-photon counting technique. Given the pulse duration of the synchrotron [l], lifetimes of the order of 20 ps must be viewed with caution, but the agreement between our value and those obtained by other workers is good enough to encourage confidence that the observed 20 ps lifetime is real. In our later measurements, the synchrotron source was sufficiently stable to allow us to collect decay data at all emission wavelengths with the excitation intensity constant. This enabled us to construct time-resolved emission spectra for the three components and these are shown in Fig. 2. All three time-resolved spectra exhibit a peak at 690 nm and the two shorter lifetime components also have a peak or shoulder at 710 nm. The positions of both of these spectral features are in agreement with the steady state spectrum, but the intensity of the longer wavelength emission appears to be rather greater than would be anticipated on the basis of the steady state spectrum. This spectral
TABLE 1 Fluorescence fritschiP
lifetimes
(ps) and percentage
Data sets analysed
h(nm)
71
Pl
Individually
680 690 700 710 720
15 fa 21+l3 23+6 26 f 5 26 f 3
64 75 80 83 85
Globally
680 690 700 710 720
22+10 22f10 22-+10 22 * 10 22-+10
55+10 71+6 81f7 8524 89f4
contributions
72
f f + f k
11 5 6 3 4
for PSI from Chlorogloea
p2
73
p3
605 515 575 660 410
+ * f * f
105 130 70 40 80
15*2 12+2 13f2 9+2 9 f 2
3500f240 3400-+380 2930f350 3610f430 3260 f 480
21 f 6 13 + 6 7k5 7+2 5f3
550 550 550 550 550
2 + + + f.
100 100 100 100 100
1723 lOf3 14f3 10+5 6 * 3
3360f400 3350+400 3350f400 3350-+400 3350 f 400
28 k 8 19 f 6 5*3 5f3 5+3
Vhe + values are the average standard deviations for the data sets analysed.
450
I
680
700 Wavelength
720 (mu)
Fig. 2. Time-resolved fluorescence ponents for PSl alone.
spectra of short (0), middle (A) and long (v) com-
feature is the subject of further investigation as we attempt to make substantial improvements in the wavelength resolution of the spectra. In addition to the fluorescence work on PSl that is summarized in Section 1, there have been a number of picosecond flash absorption studies of PSl and the kinetics of some of the component species assigned to the light-harvesting system and the reaction centre are known. Porter and coworkers [14, 151 and Il’Ina et al. [16] have reported three component kinetics for the deactivation of the antenna chlorophylls with lifetimes of 15 - 20 ps [14, 151 or 20 - 45 ps [16], approximately 300 ps and greater than 500 ps. Nuijs et al. [17] have reported a 40 + 5 ps lifetime for antenna chlorophyll decay, but this could be limited by the pulse width of their laser (full width at half maximum (FWHM), 35 ps). The excitation flash intensity used during these experiments is also variable, so any discrepancies might also be ascribed to the formation of different excited states in different proportions. We have previously reported complementary antenna decay and P,oo+ rise times of 20 ps [18], similar to the results of Porter and coworkers [14, 151. However, our most recent results [ 191 indicate that the
451
decay of the antenna pigments measured at 675 nm has a lifetime in the range 0.2 - 4.0 ps, which is difficult to resolve with our current 4 ps pulse width. This suggests that fluorescence transients ascribed to this energy transfer process may need further consideration, or that two or more lightharvesting to reaction centre energy transfer processes may be involved. Indeed, the spectral dependence of our fluorescence decay components (Fig. 2) suggests that this might be the case. All of the observed components can be assigned to fluorescence from antenna chlorophylls and complexes close to P,,. The longest-lived component of some 3.0 ns is believed to be a chlorophyll-protein complex which is completely uncoupled from the rest of the antenna and from the reaction centre. We do not believe that it is due to free chlorophyll solubilized in the detergent used in the extraction for two reasons: (i) the lifetime is a factor of two shorter than the 5.0 - 6.0 ns lifetime observed for chlorophyll a in the detergents that are used in the extraction procedure [20] and (ii) the emission is red shifted by some 10 - 20 nm from that of detergentsolubilized chlorophyll. Addition of sodium dodecyl sulphate to the PSl preparation results in a huge increase in fluorescence intensity and a decay profile which is virtually a single exponential with a lifetime of approximately 5.0 ns. Owens et al. [6,7] reached the same conclusion about the 6.0 ns component observed in PSl preparations from Chlamydomolzus reinhardtii. Our 3.0 ns lifetime is in good agreement with those assigned by Green et al. [21], Suter et al. [22] and Fleming and coworkers [23,24] to uncoupled chlorophyll. Presumably the decrease in lifetime from 6.0 to approximately 3.0 ns is due to a small amount of. residual energy transfer between the chlorophylls in the uncoupled protein complex or is an intrinsic property of chlorophyll a when complexed to a protein. In preparations from C. reinhardtii, Fleming’s group have observed three fluorescence components when the PSl preparation has a chlorophyll to P7c0 ratio of greater than 40 [6]. Concurrent with the appearance of this middle component of lifetime 250 - 2500 ps, the preparation also contains chlorophyll b. C. fritschii contains no chlorophyll b; on the basis of the similarity of the emission spectrum of the 400 - 650 ps component with that of the 3.0 ns component, we assign it to antenna complexes in which the coupling to the reaction centre has been partially disrupted. There seems little doubt that the short-lived fluorescence component is due to fluorescence from antenna complexes where energy transfer to the reaction centre is fully operational. However, based on the fast absorption changes observed at 675 nm [19], the 20 ps fluorescence cannot originate in the bulk antenna pigments. However, transient absorption changes are also observed in the wavelength region 670 - 720 nm with lifetimes of some 30 - 50 ps. These may well result from the antenna pigment complexes present in the model proposed by Wittmershaus [25] where the main antenna complexes absorbing at 680 nm (&so) and fluorescing at 690 nm pass their energy to a complex which is intermediate between them and P ,oO, This latter complex absorbs at 697 nm (Chg7) and fluoresces at 720 nm.
452
Our 20 ps component exhibits peaks at both 690 and 710 - 720 nm and we believe that this may be due to fluorescence from both C6s0 and Cbg7. The short-lived fluorescence component may be comprised of two separate decay components from these two species. Alternatively, the fluorescence from C6s0 may be derived mainly from back transfer of energy from C& and the fluorescence kinetics of C6s0 may therefore be determined by those of the C& precursor. If the lifetime values in Table 1 for the individual data analyses (rather than the global analysis) are examined, there is some evidence that they increase with wavelength. If, on the basis of the absorption data, we assign a lifetime of 0.2 - 4.0 ps to C6s0and a lifetime of 40 - 50 ps to C@T, the change in the fluorescence lifetime values as a function of wavelength could be explained by there being relatively more of the C&, fluorescence at longer wavelength. However, these very fast processes are really straining the limits of time resolution for the timecorrelated, single-photon counting technique, even with a fast laser as the excitation source. It is clear that a subpicosecond laser, together with sensitive streak camera detection, is required to probe these time scales properly. In an attempt to obtain further insight into the origin of the PSl fluorescence and the mechanisms that are operating, we added redox reagents to alter the oxidation state of P,as. In particular, we added potassium ferricyanide (6 mM) to oxidize P,aO and sodium dithionite to reduce P,oo and partially reduce the iron-sulphur acceptor complex [ 261. In both cases the lifetime of the short-lived component remains in the region of 20 ps, but the spectrum of this component varies considerably from that observed for PSl alone. The lifetime of the middle component increases to 1.2 ns but its spectrum is essentially unchanged. This is also true of the longer-lived component whose lifetime increases to 4.0 - 5.0 ns. In the case of the middle and long-lived components, it appears that the addition of the two redox chemicals further disrupts the pigment-protein complexes so that the fluorescence lifetimes increase but the spectra remain unchanged. The time-resolved fluorescence spectra of the short component of PSl with and without added potassium ferricyanide and sodium dithionite are compared in Fig. 3. The three spectra are drawn to enable comparison of the overall shapes of the spectra. The same vertical scale has not been used for each spectrum because of unquantifiable variations in excitation intensity between the three sets of data. The three spectra compared in Fig. 3 substantiate the suggestion made earlier that we are observing short-lived fluorescence from two species, both of which lie in the path of transfer of excitation energy to PToO.In the absence of any additions it is clear from Fig. 3 that the redox state of P,OOis intermediate between fully oxidized and fully reduced. When P,,,,, is reduced (in the presence of sodium dithionite), Cm0 is less fluorescent than Cbg7. This situation is reversed when PToOis oxidized. This is unexpected because if P,OOis oxidized, and therefore unable to accept
453 I
I
PSl + Dithionite
P 6130
700
720
1
I
PSl + Ferricyanide
PSl Alone
r 680 700 Wavelength
720 (nm)
680
700
Fig. 3. Time-resolved fluorescence spectra of the short-lived component presence and absence of potassium ferricyanide and sodium dithionite.
720
of PSl in the
excitation energy from C&,, the latter becomes the terminal chromophore in the energy transfer chain and should fluoresce much more strongly than when PYoo is reduced and able to accept energy. However, if C& is the terminal chromophore it will have a longer fluorescence lifetime than when it is coupled to P,,, and this may be the cause of the lengthening of the lifetime of the middle component in the presence of potassium ferricyanide. Alternatively, the ferricyanide may also oxidize C6s, and thereby cause it to be less fluorescent. It is clear that further work is needed to resolve completely the fluorescence properties of PSl. However, the evidence that we have to date supports our proposal of a species in the energy transfer chain which is intermediate between PToo and the bulk of the light-harvesting pigments [271. Acknowledgments We wish to thank the Science and Engineering Research Council and the Royal Society for financial support and Professors G. Fleming and I. Moya and Drs. B. Wittmershaus and A. Holzwarth for providing preprints of their work.
454
References
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455 18 E. H. Evans, R. Sparrow, R. G. Brown, D. Shaw, J. Barr, M. Smith and W. Toner, Fast fluorescence and absorption measurements of photosystem 1 from a cyanobacterium, Prog. Photosynth. Res., 1 (1987) 1.99 - 1.102. 19 R. W. Sparrow, E. H. Evans, R. G. Brown, M. C. W. Evans, W. Toner and R. Chittock, unpublished work. 20 R. G. Brown, E. H. Evans, S. G. Holderness, J. Manwaring and B. May, Fluorescence spectra and decay time measurements on chlorophyll a and a non-aggregating analogue, Photobiochem. Photobiophys., 5 (1983) 87 - 92. 21 B. R. Green, K. K. Karukstis and K. Sauer, Fluorescence decay kinetics of mutants of corn deficient in photosystem I and photosystem II, Biochim. Biophys. Acta, 767 (1984) 574 - 581. 22 G. W. Suter, P. Mazzola, J. Wendler and A. R. Holzwarth, Fluorescence decay kinetics in phycobilisomes from the blue-green alga Synechococcus 6301, Biochim. Biophys. Acta, 766 (1984) 269 - 276. 23 R. J. Gulotty, G. R. Fleming and R. S. Alberte, Low intensity picosecond fluorescence kinetics and excitation dynamics in barley chloroplasts, Biochim. Biophys. Acta, 682 (1982) 322 - 331. 24 R. J. Gulotty, L. Mets, R. S. Alberte and G. R. Fleming, Picosecond fluorescence study of photosynthetic mutants of Chlamydomonas reinhardtii: origin of the fluorescence decay kinetics of chloroplasts, Photochem. Photobiol., 41 (1985) 487 496. 25 B. P. Wittmershaus, Measurements and kinetic modelling of picosecond time-resolved fluorescence from photosystem I and chloroplasts, Prog. Photosynth. Res., 1 (1987) 1.75 - 1.82. 26 E. H. Evans, R. Cammack and M. C. W. Evans, Properties of the primary electron acceptor complex of photosystem I in the .blue green alga Chlbrogloea fritschii, Biochem. Biophys. Res. Commun., 68 (1976) 1212 - 1218. 27 E. H. Evans, R. Sparrow, R. G. Brown, R. Chittock, A. Langley, J. Barr, W. Toner and M. C. W. Evans, Picosecond absorption measurements of photosystem 1 from the cyanobacterium Chlorogloea fritschii, EBEC Reps., 5 (1988) 174.