Photosynthesis Research 22: 277-284, 1989. © 1989KluwerAcademicPublishers.Printedin the Netherlands. Regular paper

Energy transfer to low energy chlorophyll species prior to trapping by P700 and subsequent electron transfer D.R. Klug ~, L.B. Giorgi, B. Crystall, J. Barber* & G. Porter Photochemistry Research Group and * AFRC Photosynthesis Research Group, Department of Pure and Applied Biology, Imperial College, Prince Consort Road, London SW7 2BB, U.K.; I Authorfor correspondence Received 21 February 1989; acceptedin revisedform 22 June 1989

Key words: electron transfer, energy transfer, Photosystem One, picosecond absorption spectroscopy, primary electron acceptor A0, primary electron donor P700

Abstract It is found that the two singlet state lifetimes observed in medium sized isolated Photosystem One reaction centres belong to two distinct sets of particles. The nanosecond lifetime is due to PSI particles in which P700 does not trap excitation energy, and the excitation energy is homogeneously distributed within the antennae of these particles. The spectral features of the picosecond component show that excitation energy in the antenna has become largely concentrated in one or more low energy (red) chlorophyll species within 3.5 ps. Antennae which have become decoupled from P700 also appear to be decoupled from these red "ancillary" chlorophylls, and this suggests that some substructure or level of organisation links them to P700. The rate of quenching of antenna singlet states appears to be independent of the redox state of P700 under the conditions used here, and oxidising P700 does not prevent excitation energy from reaching the red chlorophyll species in the antenna. We find no evidence in the data presented here of a chlorophyll molecule acting as a "metastable" primary acceptor (A0). The lower limit for the detection of such a species in these data is 20% of the optical density of the transient P700 bleach. Abbreviations; Chl - chlorophyll, PS 1 - Photosystem One, P700- primary electron donor, A0- primary electron acceptor

Introduction The reaction centres of photosynthetic organisms are supplied with excitation energy by a wide variety of light harvesting complexes (Zuber 1985). The efficiency of the light harvesting process is an important factor in the viability of photosynthetic organisms. Long range energy transfer is thought to occur largely by random walks of incoherent excitons under the F6rster mechanism (Pearlstein 1982). The spectral properties of pigments engaged in light harvesting, can, in principal induce a cer-

tain degree of directionality in this process, simply by creating a negative gradient of energy levels leading to the reaction centre, but it is not clear that such a funneling of excitation energy actually occurs. The ability to perform time resolved experiments, which include some spectral information, allows directional energy transfer to be distinguished from simple heterogeneity. Although it has proved possible to isolate reaction centres from purple bacteria (Okamura et al. 1982) and higher plants (Nanba and Satoh 1987, Barber et al. 1987) which are essentially free from

278 antenna molecules, this has so far proved impossible with Photosystem 1 (PSI) reaction centres. The isolation of PS 1 reaction centres produces particles which are associated with at least two categories of antenna; the core and the peripheral antenna. The number of antenna chlorophylls associated with each particle usually varies from 30-110 (Mullet et al. 1980), although particles having as few as 7-10 chlorophyll molecules have been reported (Ikegami and Katoh 1975). It seems, however, that in its native form, the core antenna of the PSI reaction centre complex is associated with 20-50 chlorophyll-a molecules which occur in a variety of spectral forms. The nature and relevance of these spectral forms of chlorophyll is a subject for debate, as it is unclear whether they originate from a sub-population of antenna, exciton coupling, site specific effects or even artifacts of the isolation procedure. A chlorophyll molecule has also been identified as the primary electron acceptor, A0, in PS 1 reaction centres (Bonnerjea and Evans 1982). There is as yet no direct observation of the sequential reduction and oxidation of a chlorophyll molecule during charge separation. The observation o f such a species in transient absorption data would provide a useful addition to the accumulating evidence related to electron transfer reactions in proteins, particularly as the successful crystallisation of the PSI reaction centre suitable for X-ray studies (Witt et al. 1988) may eventually lead to the determination of the structure to high resolution. In this paper we report the observation of a sub-population of low energy chlorophyll-a antenna molecules which have been excited by excitation energy transfer. We also report on the role of chlorophyll as the electron acceptor A0. These observations have been made using picosecond transient absorption spectroscopy with a white light probe which allows the observation of the kinetics across 150 nm of spectrum simultaneously.

Materials and methods

The picosecond pump-probe spectrometer is o f a standard design, and produces pulses at 610nm having a 7 ps F W H M autocorrelation at 10 Hz and > 1 mJ in energy. These are split, to produce one

beam as an excitation source, while the other is focussed into a water cell to produce a white light probe. The changes in probe intensity are monitored by imaging onto a twin track vidicon which allows the collection of 150 nm of spectrum at once (Gore et al. 1986). The temporal origin of these data are taken from the midpoint of the 10% to 90% grow-in of the antenna bleach. The PS1 reaction centre isolation was performed according to the method of Alberte and Thornber (1978). P700/chlorophyll ratios were determined by chemically induced absorption changes. 1 mM ferricyanide was used to oxidise P700, and 1.5mM ascorbate to rereduce it. Although some oxidation of chlorophyll antenna molecules was observed during this procedure, this does not significantly affect the P700/chlorophyll ratios. The induced absorbance measurements assumed a differential extinction coefficient of 64 m M - ~cm- l (Hiyama and Ke 1972) for P700 at 697 nm and the chlorophyll-a content was measured in 80% acetone and with the extinction coefficient taken as 6 0 m M -~cm -~ at 677 nm (Thornber 1969). The P700/Chl ratio was 1 : 3 1 _ 2. This figure was corrected for the presence of particles lacking a functional P700, by estimating the proportion of the long lived component in the transient absorption spectra and by single photon counting (see Discussion). This yields a final figure of 25 + 5 for the average number of chlorophyll molecules in each antenna. Samples were made to an optical density of 1.4 at 673 nm in a 1 cm cuvette and stirred continuously while in the transient absorption spectrometer. The excitation intensity was adjusted so that no annihilation was observed in our data. Annihilation is observed as a component of the bleach which roughly follows the temporal profile of the excitation pulse, i.e., the bleach disappears as the intensity of the excitation pulse falls. The excitation intensity used in our experiments was such that only half of the particles absorbed any photons. This means that the probability for double excitation is only 0.15 and hence that the kinetics are not expected to show significant effects of exciton annihilation. 4 mM ascorbate was used to ensure reduction of P700 prior to flash excitation, from here on referred to as neutral P700. Preoxidised P700 was produced by adding 1 m M ferricyanide.

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Energy transfer to low energy chlorophyll species prior to trapping by P700 and subsequent electron transfer.

It is found that the two singlet state lifetimes observed in medium sized isolated Photosystem One reaction centres belong to two distinct sets of par...
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