Femtosecond transient absorption spectroscopy has been used to investigate the primary charge separation in a photosystem II deletion mutant from the cyanobacterium Synechocystis sp. PCC 6803. These cells contain only the photosystem I reaction center and have a pigment content of approximately 100 chlorophylls per P700. Utilizing relatively high excitation intensities, the difference spectrum for the reduction of primary electron acceptor [(A0(-)-A0) difference spectrum] was obtained from experiments performed under both reducing and oxidizing conditions. Both approaches yield very similar results with the (A0(-)-A0) difference spectrum displaying a maximum bleaching at 687 nm. The shape of the difference spectrum suggests that the primary electron acceptor in photosystem I may be a chlorophyll a molecule. The observed rate of primary radical pair formation depends on the overall rate of decay of excitations in the antenna; the radical pair state forms as the antenna decays. The decay of the primary radical pair state is characterized by a 21-ps time constant. Under conditions that avoid annihilation effects, the mean lifetime for excitations in the antenna is 28 ps [Hastings, G., Kleinherenbrink, F.A.M., Lin, S., & Blankenship, R.E. (1994) Biochemistry (preceding paper in this issue)]. This indicates that the reduced acceptor decays faster than it forms. Therefore, only a low concentration of the reduced acceptor will accumulate under most conditions.

Photosystem two reaction centers have been studied using a sensitive femtosecond transient absorption spectrometer. Measurements were performed at 295 K using different excitation wavelengths and excitation intensities which are shown to avoid multiphoton absorption by the reaction centers. Analyses of results collected over a range of time scales and probe wavelengths allowed the resolution of two exponential components in addition to those previously reported [Durrant, J. R., Hastings, G., Hong, Q., Barber, J., Porter, G., & Klug, D. R. (1992) Chem. Phys. Lett. 188, 54-60], plus the long-lived radical pair itself. A 21-ps component was observed. The process(es) responsible for this component was (were) found to produce bleaching of a pheophytin ground-state absorption band at 545 nm and the simultaneous appearance of a pheophytin anion absorption band at 460 nm resulting in a transient spectrum which was that of the radical pair P680+Ph-. This component is assigned to the production of reduced pheophytin. A lower limit of 60% of the final pheophytin reduction was found to occur at this rate. Despite subtle differences in transient spectra, the lifetime and yield of this pheophytin reduction are essentially independent of excitation wavelength within the signal to noise limitations of these experiments. A long-lived species was also observed. This species is produced by those processes which result in the 21-ps component, and it has a spectrum which is found to be independent of excitation wavelength. This spectrum is characteristic of the primary radical pair state P680+Ph-. In addition, a 200-ps component was found which is tentatively assigned to a slow energy-transfer/trapping process. This component was absent if P680 was excited directly and is therefore not integral to primary radical pair formation. Overall, it is concluded that the rate of pheophytin reduction is limited to (21 ps)-1, even when P680 is directly excited.

PhotosystemIH reaction centers have been studied by femtoend trent absorption spectroseopy. We demonstrate that it is possible to achieve good photoselectivity between the primary electron donor P680 and the majority of the accessory chlorins. Energy tanser can be observed in both directions between P680 and these accessory chlorins depending on which Is initily excited. This oxidizing potential is used to drive water splitting, which gives rise to oxygen evolution. The primary electron donor of PSII is thought to correspond to a spectral feature at 680 nm and is referred to as P680 (3), while a pheophytin (Ph) molecule functions as an electron acceptor (4-6). Studies of PSII core complexes binding 60 and 80 antenna chlorophylls have suggested that the primary radical pair P680+Ph-is formed at a rate of 4100 psfollowing the absorption ofa photon by antenna pigments (7).A similar conclusion was reached from photovoltage studies of larger PSII complexes (8). A kinetic model, in which there is a rapid (--1 ps) equilibration of excitation energy between the antenna pigments and P680, followed by the observed trapping of the excitation energy by radical pair formation in ""100 ps, has been proposed for this process (refs. 9 and 10; reviewed in ref. 2). This so-called trapping limited model (11) is valid when the rate of electron transfer from the primary electron donor is slower than energy transfer back to the antenna pigments. This model has also been applied to other photosynthetic antenna/reaction center complexes (12)(13)(14).However, previous studies have not been able to time resolve the energy-transfer processes that are predicted to cause the equilibration of excitation energy between the antenna and primary electron donor pigments prior to radical pair formation.We report here a study ofexcitation energy equilibration in the isolated Dl/D2/cytochrome b559 complex, which is the reaction center of PSII. This complex is much smaller than the isolated PSII core complexes discussed above, binding only six chlorophyll a and two Ph a pigments (15, 16). While several of these pigments are presumably involved in electron-transfer processes, these pigments will also function in an energy-transferring capacity before charge separation. Time-resolved fluorescence studies have determined that at least 94% of the chlorin pigments in our PSI1 reaction center preparation are able to transfer excitation energy to P680, resulting in a near unity quantum yield of the primary radical pair (17,18). In a previous study, we determined that when P680 is directly excited, Ph reduction occurs primarily at a rate of 21 ps-1 in isolated PSII reaction centers at room temperature (4).There have been several discussions of the similarities between the PSII reaction center of higher plants and the reaction center of purple bacteria (19,20). However, these two complexes are likely to be very different in terms oftheir energy-transfer kinetics when isolated from their antenna systems. The lowest SO-S, opticil transition for the pr...

Picosecond fluorescence and femtosecond transient absorption spectroscopy have been used to investigate the primary energy transfer and trapping processes in a photosystem II deletion mutant from the cyanobacterium Synechocystis sp. PCC 6803, which contains active photosystem I reaction centers with approximately 100 chlorophylls per P700. In all experiments, low levels of excitation were used which avoid annihilation processes. Following 590-nm excitation, at room temperature, spectral equilibration is observed in both fluorescence and absorption measurements and is characterized by a time constant of 4-6 ps. The shape of the spectra associated with the equilibration process indicates that long wavelength pigments (pigments with absorption maxima at longer wavelength than that of the primary electron donor, P700) are present and functional at physiological temperatures in this preparation. The overall decay of excitations in the antenna is characterized by a time constant of 24-28 ps, in both fluorescence and absorption measurements. The 24-28-ps process results in the appearance of absorption changes associated with only P700+ formation. Absorption changes associated with the reduction of the primary electron acceptor were not resolved under the experimental conditions used here.

Femtosecond transient absorption spectroscopy has been used to investigate the photoinduced energy and electron transfer processes in photosystem I (PS I) particles from cyanobacteria, green algae, and higher plants. At room temperature, the kinetics observed in all three species are very similar: Following 590 nm excitation, an equilibration process(es) with a 3.7-7.5 ps lifetime was observed, followed by a 19-24 ps process that is associated with trapping. In all three species long-wavelength pigments (pigments that absorb at longer wavelengths than the primary electron donor) were observed. The difference spectrum associated with reduction of the primary electron acceptor [Ao(-)-Ao) difference spectrum] was obtained for all three species. The (Ao(-)-Ao) difference spectra obtained from measurements using detergent-isolated PS I particles from spinach and Chlamydomonas reinhardtii are similar but clearly membrane fragments. In all three species the reduced primary electron acceptor (Ao(-)) is reoxidized extremely rapidly, in about 20 ps. The difference spectrum associated with Ao reduction appears to contain contributions from more than a single chlorophyll pigment.