The energy transfer and charge separation kinetics of a photosystem I (PS I) core particle of an antenna size of 100 chlorophyll/P700 has been studied by combined fluorescence and transient absorption kinetics with picosecond resolution. This is the first combined picosecond study of transient absorption and fluorescence carried out on a PS I particle and the results are consistent with each other. The data were analyzed by both global lifetime and global target analysis procedures. In fluorescence major lifetime components were found to be 12 and 36 ps. The shorter-lived one shows a negative amplitude at long wavelengths and is attributed to an energy transfer process between pigments in the main antenna Chl pool and a small long-wavelength Chl pool emitting around 720 nm whereas the longer-lived component is assigned to the overall charge separation lifetime. The lifetimes resolved in transient absorption are 7-8 ps, 33 ps, and [unk]1 ns. The shortest-lived one is assigned to energy transfer between the same pigment pools as observed also in fluorescence kinetics, the middle component of 33 ps to the overall charge separation, and the long-lived component to the lifetime of the oxidized primary donor P700(+). The transient absorption data indicate an even faster, but kinetically unresolved energy transfer component in the main Chl pool with a lifetime <3 ps. Several kinetic models were tested on both the fluorescence and the picosecond absorption data by global target analysis procedures. A model where the long-wave pigments are spatially and kinetically connected with the reaction center P700 is favored over a model where P700 is connected more closely with the main Chl pool. Our data show that the charge separation kinetics in these PS I particles is essentially trap limited. The relevance of our data with respect to other time-resolved studies on PS I core particles is discussed, in particular with respect to the nature and function of the long-wave pigments. From the transient absorption data we do not see any evidence for the occurrence of a reduced Chl primary electron acceptor, but we also can not exclude that possibility, provided that reoxidation of that acceptor should occur within a time <40 ps.

A silicon single-photon avalanche diode (SPAD) detector and a proximity-focus-type microchannel-plate photomultiplier tube (MCP-PMT) are compared with respect to their performance in time-correlated single-photon counting (TCSPC) applications. A solution of pinacyanol (1,1′-diethyl–2,2′-carbocyanine) in ethanol, which is used as a test substance with short-lived fluorescence, is excited at 620 nm with a synchronously pumped, cavity-dumped rhodamine 6G dye laser. Deconvolution analysis of the fluorescence decay with a biexponential decay model gives excellent fits to the experimental data recorded with any one of the detectors. The fluorescence lifetimes for the dominating decay component are 13±2 and 11±2 ps as measured at 635 nm with the SPAD detector and the MCP-PMT, respectively. The excellent agreement of these values and the quality of the fits show that the SPAD detector as well as the MCP-PMT can resolve fluorescence decay time constants of the order of 10 ps. This is significantly better than what had been expected on the basis of the instrumental response widths of 70 ps (FWHM) with the SPAD detector and 50 ps (FWHM) with the MCP-PMT.

A detailed model for the kinetics and energetics of the exciton trapping, charge separation, charge recombination, and charge stabilization processes in photosystem (PS) II is presented. The rate constants describing these processes in open and closed reaction centers (RC) are calculated on the basis of picosecond data (Schatz, G. H., H. Brock, and A. R. Holzwarth. 1987. Proc. Natl. Acad. Sci. USA. 84:8414-8418) obtained for oxygen-evolving PS II particles from Synechococcus sp. with approximately 80 chlorophylls/P(680). The analysis gives the following results. (a) The PS II reaction center donor chlorophyll P(680) constitutes a shallow trap, and charge separation is overall trap limited. (b) The rate constant of charge separation drops by a factor of approximately 6 when going from open (Q-oxidized) to closed (Q-reduced) reaction centers. Thus the redox state of Q controls the yield of radical pair formation and the exciton lifetime in the Chl antenna. (c) The intrinsic rate constant of charge separation in open PS II reaction centers is calculated to be approximately 2.7 ps(-1). (d) In particles with open RC the charge separation step is exergonic with a decrease in standard free energy of approximately 38 meV. (e) In particles with closed RC the radical pair formation is endergonic by approximately 12 meV. We conclude on the basis of these results that the long-lived (nanoseconds) fluorescence generally observed with closed PS II reaction centers is prompt fluorescence and that the amount of primary radical pair formation is decreased significantly upon closing of the RC.

Oxygen-evolving photosystem II particles (from Synechococcus) with about 80 chlorophyll molecules per primary electron donor (P(680)) were used for a correlated study of picosecond kinetics of fluorescence and absorbance changes, detected by the single-photon-timing technique and by a pump-probe apparatus, respectively. Chlorophyll fluorescence decays were biexponential with lifetimes tau(1) = 80 +/- 20 ps and tau(2) = 520 +/- 120 ps in open reaction centers and tau(1) = 220 +/- 30 ps and tau(2) = 1.3 +/- 0.15 ns in closed reaction centers. The corresponding fluorescence yield ratio F(max)/F(o) was 3-4. Absorbance changes were monitored in the spectral range of 620-700 nm after excitation at 675 nm with 10-ps pulses sufficiently weak (<7 x 10(12) photons/cm(2) per pulse) to avoid singlet-singlet annihilation. With open reaction centers, the absorbance changes could be fit to the sum of three exponentials. The associated absorbance difference spectra were attributed to (i) exciton trapping and charge separation (tau = 100 +/- 20 ps), (ii) the electron-transfer step P(680) (+) I(-) Q(A) --> P(680) (+) I Q(A) (-) (where I is the primary electron acceptor and Q(A) is the first quinone acceptor) (tau = 510 +/- 50 ps), and (iii) the reduction of P(680) (+) by the intact donor side (tau > 10 ns). With closed reaction centers, the absorbance changes were biexponential with lifetimes tau(1) = 170-260 ps and tau(2) = 1.6-1.75 ns. The results are explained in terms of a kinetic model that assumes P(680) to constitute a shallow trap. The results show that Q(A) reduction in these photosystem II particles decreases both the apparent rate and the yield of the primary charge separation by a factor of 2-3 and increases the mean lifetime of excitons in the antenna by a factor of 3-4. Thus, we conclude that the long-lived, nanosecond chlorophyll fluorescence is not charge-recombination luminescence but rather emission from equilibrated excited states of antenna chlorophylls.

The supramolecular structure of the exoplasmic freeze-fracture particles of thylakoids of the thermophilic cyanobacterium Synechococcus sp. is compared with that of isolated photosystem-II complexes. The in-situ EF particles are scattered on the thylakoids or organized in rows of variable length; the latter aligned particles measure 10 nmx20 nm and are separated perpendicular to their long axis into two parts. We propose that they represent dimers composed of two monomeric 10-nm EF particles side by side. Isolated photosystem (PS)II particles correspond in size to the monomeric 10-nm EF particles as analysed by negative contrast and freeze-fracture electron microscopy. Dimeric PSII particles, very similar to the in-situ 10 nmx20 nm EF particles, are obtained after incorporation of purified PSII complexes into liposomes made from phospholipid and cholesterol. Each monomeric complex consists of the reaction center, the water-splitting system, the chlorophyll antennae and phycobilisome-binding polypeptides. We propose that the dimeric complexes bind one hemidiscoidal phycobilisome at their domains exposed to the external side of the thylakoids. The implications of this arrangement of the PSII-phycobilisome complexes within the thylakoids upon excitation-energy distribution are discussed.

Charge-transfer reactions to secondary electron donors (Z, M) and acceptors (QA, QB) in Photosystem II particles isolated from a thermophilic cyanobacterium Synechococcus sp. (Schatz, G.H. and Witt H.T. (1984) Photobiochem. Photobiophys. 7, 1–14) were analyzed by measurements of fluorescence yield and absorbance changes in the millisecond time domain induced by repetitive flashes. (1) The electron-transfer reaction Q−AQB → QAQ−B was found to occur with kinetic phases of 0.2 ± 0.1 ms and 1.5 ± 0.5 ms half-time. At 10 ms after flashes an equilibrium distribution of Q−AQBQAQ−B of about 1585 in oxygen-evolving and of about 2575 in Tris-treated PS II particles was reached. (2) The absorbance difference spectra were determined for (Q−A - QA), (Q−B - QB), (Z+ - Z) and for (S4 - S0), the transition associated with oxygen evolution. In the ultraviolet region they show that these electron-acceptors and -donors are the same as in spinach PS II. In the visible region all the difference spectra contain major contributions by electrochromic bandshifts due to electrostatic interaction of the reduced acceptors or oxidized donors with nearby reaction center pigments. Upon electron transfer from Q−Ato QB electrochromic bandshifts due to interaction with pheophytin a disappeared almost completely. Bandshifts observed in the (Z+ - Z) and (S4 - S0) spectra were attributed to chlorophyll a.