Determination of P680 singlet state lifetimes in photosystem two reaction centres

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3 January 1992

Determination of P680 singlet state lifetimes in photosystem two reaction centres James R. Durrant a*b,Gary Hastings a, Qiang Hong a, James Barber b, George Porter a and David R. Klug a a Photochemistry Research Group. Department of Biology, Imperial College, London SW7 ZBB, UK b AFRC Photosynthesrs Research Group, Department ofBiochemistry,Imperial College.London SW7 2BB, UK Received 20 September I99 I

Photosystem two reaction centres have been studied using femtosecond transient absorption spectroscopy, employing sufflciently low excitation levels to avoid multiphoton processes. The absorption changes are shown to be strongly dependent on excitation wavelength. Transiently bleached P680 is found to have a higher oscillator strength than monomeric chlorophyll molccules, suggesting that the ~680 excited singlet state is initially delocalised. When P680 is directly excited, its singlet excited state is observed to decay with lifetimes of 400+ 100 fs and 3.5 k 1.5 ps.

1. Introduction Photosynthetic reaction centres are chromophore-protein complexes which convert absorbed photons into an electrochemical potential. This potential is formed by sequential electron transfer reactions which occur with quantum yields of x 1. The study of higher plant reactions centres has been hampered until recently by the absence of appropriate isolation procedures, and this has meant that studies of the first electron transfer reactions, normally referred to as primary charge separation, have largely been confined to the reaction centres of purple bacteria such as Rhodobacter sphaeroides and Rhodopseudomonas Widis. Although reaction centres from purple bacteria were first isolated 23 years ago [ 11, and despite the solution of their crystal structures, the precise mechanism of primary radical pair formation remains the subject of much contention [ 2-

41. The photosystem two (PS2) reaction centre of higher plants is of particular interest due to its ability to produce an unusually high oxidising potential of x 1 V. This oxidising potential is used to extract electrons from water and release O2 via a series of reactions which occur within a manganese cluster closely associated with the oxidising side of the re54

action centre. Thus although the major polypeptides of the PS2 reaction centre are homologous with those of purple bacteria, some modifications must exist to allow it to drive water splitting. The isolation and identification of a PS2 reaction centre [ $61 has provided an opportunity to study primary electron transfer in a higher plant system, without the complications caused by chlorophyll antenna complexes and secondary quinone electron acceptors. Studies of the isolated PS2 reaction centre were initially restricted by its relative lability. It is, however, now possible [ 7,8,12] to produce highly functional and stabilised PS2 reaction centres. These reaction centres retain their ability to carry out primary charge separation, resulting in the formation of the primary radical pair state, P680+Ph- [9-l 11, with a near unity quantum yield, even when exposed to fairly high light levels for periods of up to an hour [121. Transient absorption studies of PS2 reaction centres are hampered by the extensive overlap of spectral features. This means that very accurate data are required in order to distinguish and identify the energy and electron transfers which follow the absorption of light. In particular it is necessary to keep excitation levels low to avoid multiphoton processes, and to investigate the effects of varying excitation

0009-2614/92/$ 05.00 Q 1992 Elsevier Science Publishers B.V. All rights reserved.

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wavelength. In this Letter we report experiments performed with an appropriately designed femtosecond transient absorption spectrometer, and demonstrate that important features related to the primary charge separation in PS2 can be observed.

2. Materials and methods Reaction centres were isolated from pea thylakoid membranes and resuspended as in previous measurements [ 121. Anaerobic conditions were achieved as previously [ 8 1. All transient absorption measurements were performed at 295 K in a 2.5 mm path length cuvette which was rotated at sufficient speed to replace the sample volume between flashes. The optical densities of the samples at the peak of the longest wavelength absorption band (675.5 nm) were between 0.8 and 1.0. Samples were exposed to light from the spectrometer for approximately 1 h, during which the peak of the long wavelength absorption band shifted by less than 1 nm, corresponding to less than a lOohloss in activity [ 121. The observed absorption changes were found to be the same, within signal to noise, at the beginning and end of 1 h exposure to light in the apparatus. The apparatus used to conduct the transient absorption experiments will be described in detail elsewhere. PS2 reaction centres were excited using 0.1 uJ pulses centred at either 612 or 694 nm, at a repetition rate of 6.5 kHz, while the resulting changes in optical density were monitored using white light probe pulses. Excitation and probe beams were parallel polarised to better than 90%, with a 275 pm beam diameter at the sample. The time resolution ( 1O-90% response time) of the spectrometer was 160 fs, determined initially by autocorrelation of the pulses, and then routinely by monitoring the IO-90% rise time of absorption changes observed in three dye standards over a wide range of probe wavelengths. The 6 12 nm excitation pulses were within a factor of two of the transform limit ( 10 nm bandwidth ). The 694 nm pulses had a much broader spectrum (24 nm bandwidth), although only part of this pulse spectrum overlapped the reaction centre absorption spectrum. Absorption changes were monitored as a function of time at single wavelengths between 655 and 695

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nm (detection bandwidth 2 nm). Data presented in this paper were collected over a O-13 ps timescale, with a time delay of 66 fs between points, The analysis of data collected on longer timescales is presented elsewhere [ 131, with a total of over 1000 decays collected and analysed. Twelve decays were collected with each sample, with 5 min of signal averaging for each decay. The data were analysed assuming multiexponential kinetics as described by Hastings et al. [ 13 1, with decays being analysed both individually and globally, The quality of the fits was assessed using a reduced x2 criterion and plots of weighted residuals. The lifetimes of kinetic components were determined by the simultaneous global analysis of data collected at up to twelve wavelengths. Reproducibility was determined from global analyses of data from different samples, and is quoted as one standard deviation. Zero time delay was taken as the centre of the growin of the initial absorption change. Data were fitted only for times after 180 fs, and kinetic spectra obtained from the spectra of the pre-exponential factors by extrapolation back to zero time. Estimations of the excitation levels both from the pump beam parameters, and independently from the size of the transient bleaches, suggested that only 510%of the reaction centres in the volume of the pump beam were excited by each flash. In addition, we performed a saturation study which determined that both identical kinetics and identical ratios of amplitudes (within our signal to noise) were observed for all components using excitation pulse energies attenuated by up to a factor of 8. This indicates that the effects of multiphoton processes are negligible for these data due to the use of low excitation intensities.

3. Results Transient absorption data were collected between 655 and 695 nm using either 612 or 694 nm excitation pulses, Fig. 1 shows some typical data. Excitation at 694 nm rather than 612 nm results in an approximately fivefold increase in the amplitude of sub-10 ps kinetic components, as is shown in fig. 1 at ~682 nm. The absorption changes observed following excitation at 612 nm are well fit over the spectral range 55

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ing component to fit the data satisfactorily at all wavelengths between 655 and 690 nm, and at time delays up to 400 ps. The slowest exponential component was included as fixed component with a lifetime of 2 1 ps on the basis of the analysis of data obtained on longer time scales 1131, The lifetimes of the fastest components are 400 t 100 fs and 3.5 f 1.5 ps. The kinetic spectra (sometimes called decay associated spectra) of these two components and the non-decaying component are shown in fig. 2. Fig. 3a shows the difference spectra obtained fol-

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Fig. I. The kinetics of the transient absorption change observed (a) at 683 nm following excitation at 612 nm, (b) at 682 nm following excitation al 694 nm, and (c) at 660 nm following excitation at 694 nm. The solid lines are the best tits to the data with (a) two exponential components with lifetimes of I.6 and 21 ps and a non-decaying component, and (b) and (c) three exponential components with lifetimes of 400 fs, 3.5 and 2 1 ps and a non-decaying component. The lifetimes of the exponential components were determined from global analyses of data obtained over a range of time scales.

660-695 nm by the sum of two exponentials, with lifetimes of 1.6kO.6 and 21-+_3ps, plus a non-decaying (lifetime B 300 ps) component. Analysis of the 2 I ps component is presented elsewhere [ 131, and its lifetime and spectral dependence were included as fixed parameters in the analysis of all the data presented here, The kinetics of the absorption changes observed following excitation at 694 nm requires a minimum of three exponential components and a non-decay56

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wavelength(nm) Fig. 2. Kinetic spectra of the transient absorption changes observed following excitation at 694 nm: 400 fs component (O), 3.5 ps component (e) and a nondecaying component ( x ). The spectra of both the 400 fs and 3.5 ps components have negative maxima at approximately 682 ntn, corresponding to a recovery of the initial negative absorption change, whilst the positive feature in the spectrum of the 400 fs component between 660 and 675 nm may correspond to an increased bleaching of a shorter wavelength ground state absorption band. Data collected between 655 and 670 nm have significantly smaller error bars, because these data are less affected by laser scatter from the 694 nm excitation pulses and noise which is proportional lo signal amplitude.

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The 1.6 ps component of relatively small amplitude, which is observed following excitation at 612 nm, may be the mean of the two fast components which are observed following excitation at 694 nm. Their total amplitude is too small to allow them to be distinguished when 6 12 nm excitation is used. The 400 fs component is easily observed at probe wavelengths far from the excitation wavelength (see fig. 1 ), and therefore cannot be attributed to a coherent coupling artefact. A coherent optical interaction in the windows of the glass cuvette was found to distort some data for time delays less than 180 fs. In order to avoid this artifact contributing to our analysis, data were analysed starting from 180 fs. The lifetimes and spectra of the faster components recovered from our analysis were found to be only weakly affected by the presence of the 2 1 ps component.

4. Discussion

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Fig. 3. Spectra of (a) the absorption change at zero time delay obtained by extrapolation of the best fits to the observed transient absorption data back to zero time, and (b) the non-decaying component (lifetime B 300 ps) assigned to the primary radical pair state, following excitation at 612 nm (0) and 694 nm ( l ) . These spectra were obtained from data collected over a range oftime scales. The spectra obtained using 694 nm excitation were increased in amplitudeby a factorof 1.2 (see text).

lowing excitation at 612 and 694 nm by extrapolation of the kinetics back to zero time. These “initial” absorption difference spectra do not include contributions from any kinetic components faster than our time resolution of 160 fs. Fig. 3b shows two spectra of a kinetic component which is non-decaying over 300 ps; the spectrum of this component is clearly independent of excitation wavelength. This non-decaying component is assigned to the primary radical pair state P680fPh- [ 9,10,13], with a lifetime of tens of nanoseconds [ II,1 2,14 1. Excitation at 694 nm rather than 612 nm clearly results in the initial absorption difference spectrum (fig. 3a) being red shifted, narrower (fwhm bandwidth of 13 nm compared to 16 nm ) and approximately 1.9 times greater in amplitude. These spectra were normal&d by scaling by the ratio of the final radical pair spectra, as is discussed below.

Excitation of PS2 reaction centres produces the primary radical pair state P680+Ph- [ 1l] with a quantum yield near unity [ 121. The term P680 refers to the chromophore(s) responsible for the spectral feature observed at 680 nm in studies of PS2 [ 151. These chromophore( s) probably constitute the primary electron donor of PS2. 694 nm excitation pulses were used with the intention of directly exciting P680 in a much higher proportion of reaction centres than is achieved using 612 nm excitation pulses. Complete selectivity is not possible due to the high degree of spectral overlap between individual chromophores in the PS2 reaction centre, and the finite width of the excitation pulse. The initial transient absorption change observed using the 694 nm excitation (fig. 3a, closed symbols) has a similar peak (682? 1 nm) and bandwidth ( I3 & I nm) to those previously observed for the P680 Q,-absorption band ( [ 16-181 and references therein ), but extends to longer wavelengths (cf. for example fig. 3b) indicating the presence of stimulated emission. For excited singlet states, the oscillator strengths for ground state absorption and stimulated emission are expected to be of similar magnitude, with the stimulated emission generally extending to longer wavelengths relative to the ground state bleach. The initial difference spectrum 57

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observed following excitation at 694 nm (fig. 3a) is therefore assigned primarily to the P680 excited singlet state, although a limited contribution from other chlorin singlet states cannot be excluded. Excitation at 612 nm results in the bleaching of a significant proportion of shorter wavelength absorbing chromophores (fig. 3a open symbols); this spectrum also exhibits the presence of stimulated emission. Fig. 3 therefore demonstrates that the excitation energy does not fully equilibrate between the chromophores of the PS2 reaction centre within the first 180 fs. It can be concluded that excitation at 694 nm rather than 612 nm results in a relatively selective excitation of P680, and that this selectivity is retained for the first 180 fs. Excitation at 694 nm also results in the initial absorption change being almost twice that observed following excitation at 612 nm (fig. 3a), when the spectra are normalised to give the same final radical pair yield. This normalisation takes account of variations in excited state concentration and orientation of the reaction centres when using the different excitation pulses. It can readily be estimated that the total radical pair quantum yield is independent of excitation wavelength to within + 10%. (In order to account for the observed difference in the amplitudes of the spectra shown in fig. 3a, 694 nm excitation would have to result in a 50% lower radical pair quantum yield than 612 nm excitation; this is not only implausible but also inconsistent with timeresolved fluorescence data obtained using variable wavelength excitation [ 191.) Both spectra shown in fig. 3a are assigned to chlorin singlet excited states, and it is unlikely that the observed difference between the spectra could be attributed to differences in excited state absorption. The simplest explanation of this difference in the initial absorption change is that excitation at 694 nm rather than 6 12 nm excites molecules which exhibit significantly higher Q,band oscillator strengths, presumably resulting from a more delocalised singlet state. As excitation at 694 nm rather than 6 I2 nm appears to result in the excitation energy residing to a greater degree upon P680, this suggests that the singlet state of P680 is to some degree delocalised over at least two coupled chlorin molecules. Our conclusion that P680 comprises at least two coupled chlorin molecules is supported by absorption spectroscopy of the P680 triplet state 58

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[ 17,20,21], circular dicbroism [ 18,22 ] and Gaussian deconvolution [ 18,211 studies of PS2 reaction centres. This conclusion is consistent with the suggestion, made primarily on the basis of amino acid sequence homologies, that P680 is a pair of chlorophyll molecules analogous to the special pair found in the reaction centres of purple bacteria [ 23,241 (see also van Mieghem et al. [25] for an alternative suggestion). The nature of P680 is the subject of some debate [25,26], and it is certainly possible that the P680 singlet state can be delocalised, whilst the triplet (and possibly cation) states of P680 may be more localised [ 17,18,20,21,26]. The 400 fs and 3.5 ps components observed following excitation at 694 nm result in an approximately 50% loss of the initial absorption change between 670 and 690 nm. It follows from the discussion above, that this recovery must originate from a loss of P680 ground state bleach and/or stimulated emission, and these components are therefore assigned to the decay of the P680 excited singlet state (P680*). It is, however, more difficult to determine the product state or states which result from these processes. Decay of a delocalised P680 singlet state to one which is more weakly coupled to the ground state, such as P680+ or an intradimer charge transfer state, would result in a reduction of stimulated emission. The recovery of the initial absorption change could in part be caused by localisation of the P680* state. Alternatively the observed recovery may be caused by energy transfer which results in equilibration of the excitation energy between the chlorins in the reaction centre. This would result in a net flow of excitation energy away from P680 due to the proximity of the S, energy levels of the other chlorin molecules associated with the reaction centre, and a net reduction in the magnitude of both the ground state bleach and stimulated emission if the P680 oscillator strength were greater in magnitude than the other chlorins. Although they are spectrally similar, both the 400 fs and 3.5 ps components are required in order to obtain reasonable fits to the globally analysed data. These components may result from two different processes. Alternatively they may both originate from the same electron transfer, energy transfer or relaxation process, with this process exhibiting some variability in its rate. It has recently been suggested that

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there is a distribution of electron transfer rates in reaction centres of purple bacteria [ 31, although an alternative model has also been suggested [ 2 1. Transient hole burning experiments have concluded that the lifetime of the P680* state is 1.9 + 0.2 ps at 4 K [ 27 ] ; however, the higher temperature (295 K) used in our study prevents a direct comparison with this observation_ Excitation at 6 12 nm results in a five times smaller amplitude (between 670 and 690 nm) for the fast kinetic components, but a similar final radical pair yield. This is consistent with the conclusion reached above that excitation at 612 nm does not allow the trapping of all excitation energy by P680 within the first few hundred femtoseconds, and with the assignment of the 400 fs and 3.5 ps components observed following excitation at 694 nm to the decay of the P680 singlet excited state. Studies on longer timescales have shown that some energy transfer/ trapping kinetics can be observed with a lifetime as long as 210 ps [ 131 when 612 nm excitation is used. Previous transient absorption studies have reported a lifetime of 2.6 ps for the state P680* [28]. It should be noted that the data presented here show kinetic changes of opposite sign at 680 nm to those observed by Wasielewski et al. [28] and it must be concluded that the kinetic process observed by Wasielewski et al. has a different origin to the processes observed in this paper. The results presented in this paper show that the primary electron donor state of PS2, P680*, decays with lifetimes of 400 fs and 3.5 ps when P6SO is directly excited. This P6SO singlet state appears to be delocalised over at least two chlorin molecules. Studies on longer time scales show that at least 50% of the pheophytin reduction observed following excitation of PS2 RCs at 694 nm occurs with a lifetime of 21 ps [ 131. These observations must be combined, and further assignments of these processes made, in order to produce a complete and testable kinetic model. The results presented here and by Hastings et al. [ 13] demonstrate that this is a realistic goal for PS2 reaction centres.

Acknowledgement

We would like to thank Niall Walsh and Caroline

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Woollin for preparing the reaction centre samples and Chris Barnett for excellent technical assistance. Thanks to Linda Giorgi for her helpful comments, and to Oxford Lasers for loan of the copper vapour laser during the early stages of this work. We also acknowledge financial support from the Science and Engineering Research Council, the Agriculture and Food Research Council and The Royal Society.

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[ 22 ] W.-Z. He, A. Telfer, A. Drake, J. Hoadley and J. Barber, in: Current research in photosynthesis, Vol. I, ed. M. Baltscheffsky (Kluwer, Dordrecht, 1990) pp. 431-434. [ 231 H. Michel and J. Deisenhofer, Biochemistry 27 ( 1988) I. [ 241 J. Barber, Trends in Biochem. Sci. I2 ( 1987) 321. [25]F.J.E. van Mieghem, W. Nitschke, P. Mathis and A.W. Rutherford, Biochim. Biophys. Acta 977 (1989) 207.

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[26] A.W. Rutherford, Biochem. Sot. Trans. 14 ( 1986) 15. [27] R. Jankowiak, D. Tang, G.J. Small and M. Seibert, J. Phys. Chem. 93 (1989) 1649. [28] M.R. Wasielewski, D.G. Johnson, M. Seibert and Govindjee, Proc. Natl. Acad. Sci. US 86 (1989) 524.