Primary Radical Pair Formation Research Articles

Charge Separation is Virtually Irreversible in Photosystem II Core Complexes with Oxidized Primary Quinone Acceptor

X-ray structures of the Photosystem II (PSII) core revealed relatively large interpigment distances between the CP43 and CP47 antenna complexes and the reaction center (RC) with respect to the interpigment distances in a single unit. This finding questions the possibility of fast energy equilibration among the antenna and the RC, which has been the basic explanation for the measured PSII fluorescence kinetics for more than two decades. In this study, we present time-resolved fluorescence measurements obtained with a streak-camera setup on PSII core complexes from Thermosynechococcus elongatus at room temperature (RT) and at 77 K. Kinetic modeling of the RT data obtained with oxidized quinone acceptor Q(A), reveals that the kinetics are best described by fast primary charge separation at a time scale of 1.5 ps and slow energy transfer from the antenna into the RC, which results in an energy equilibration time between the antenna and the RC of about 44 ps. This model is consistent with structure-based computations. Primary radical pair formation was found to be a virtually irreversible process. Energy equilibration within the CP43 and CP47 complexes is shown to occur at a time scale of 8 ps. Kinetic modeling of the 77 K data reveals similar energy transfer time scales in the antenna units and among the antenna and the RC as at RT, respectively, 7 and 37 ps. We conclude that the energy transfer from the CP43/CP47 antenna to the RC is the dominant factor in the total charge separation kinetics in intact PSII cores.

Ultrafast Excited-State Dynamics and Photolysis in Base-Off B12 Coenzymes and Analogues: Absence of the trans-Nitrogenous Ligand Opens a Channel for Rapid Nonradiative Decay

Ultrafast transient absorption spectroscopy was used to investigate the photochemistry of adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), and n-propylcobalamin (PrCbl) at pH 2 where the axial nitrogenous ligand is replaced by a water molecule. The evolution of the difference spectrum reveals the internal conversion process and spectral characteristics of the S(1) excited state. The photolysis yield in the base-off cobalamins is controlled by competition between internal conversion and bond homolysis. This is in direct contrast to the process in most base-on alkylcobalamins where primary photolysis occurs with near unit quantum yield and the photolysis yield is controlled by competition between diffusive separation of the radical pair and geminate recombination. The absence of the axial nitrogenous ligand in the base-off cobalamins modifies the electronic structure and opens a channel for fast nonradiative decay. This channel competes effectively with the channel for bond dissociation, dropping the quantum yield for primary radical pair formation from unity in base-on PrCbl and AdoCbl to 0.2 ± 0.1 and 0.12 ± 0.06 in base-off PrCbl and AdoCbl, respectively. The photolysis of base-off MeCbl is similar to that of base-off AdoCbl and PrCbl with competition between rapid nonradiative decay leading to ground state recovery and formation of a radical pair following bond homolysis.

Modulation of quantum yield of primary radical pair formation in photosystem II by site-directed mutagenesis affecting radical cations and anions.

Pigment-protein interactions play a significant role in determining the properties of photosynthetic complexes. Site-directed mutants of Synechocystis PCC 6803 have been prepared which modify the redox potential of the primary radical pair anion and cation. In one set of mutants, the environment of P680, the primary electron donor of Photosystem II, has been modified by altering the residue at D1-His198. It has been proposed that this residue is an axial ligand to the magnesium cation. In the other set, the D1-Gln130 residue, which is thought to interact with the C9-keto group of the pheophytin electron acceptor, has been changed. The effect of these mutations is to alter the free energy of the primary radical pair state, which causes a change in the equilibrium between excited singlet states and radical pair states. We show that the free energy of the primary radical pair can be increased or decreased by modifications at either the D1-His198 or the D1-Gln130 sites. This is demonstrated by using three independent measures of quantum yield and equilibrium constant, which exhibit a quantitative correlation. These data also indicate the presence of a fast nonradiative decay pathway that competes with primary charge separation. These results emphasize the sensitivity of the primary processes of PS II to small changes in the free energy of the primary radical pair.

Cu(II)-inhibitory effect on photosystem II from higher plants. A picosecond time-resolved fluorescence study.

The influence of Cu(II) inhibition on the primary reactions of photosystem II (PSII) electron transport was studied by picosecond time-resolved fluorescence on isolated PSII membranes. The fluorescence decay from Cu(II)-inhibited PSII centers showed a dominant amplitude of a fast phase (100-300 ps) similar to PSII centers in the uninhibited "open state" and minor contributions of components around 600 ps and 2.6 ns. These data indicate efficient primary charge separation in PSII membranes incubated with Cu(II). The quantum yield of primary reactions in the inhibited PSII centers was similar to that of "open" PSII centers. Kinetic analysis of the decay curves in the framework of the exciton/radical pair equilibrium model showed no significant changes in the rate constants associated with the charge separation/recombination equilibrium. However, in closed centers (QA reduced), a decrease in the rate constant K23, associated with the back-reaction of a relaxed radical pair, by a factor of 4 was calculated. The free energy losses upon primary charge separation (delta G1) and during subsequent radical pair relaxation (delta G2) were also determined in Cu(II)-inhibited centers and were compared with uninhibited centers. No changes in the delta G1 values and a significant decrease in the delta G2 values were found as compared with those of control PSII centers in the "closed" state. These data indicate that Cu(II) does not affect primary radical pair formation, but strongly affects the formation of a relaxed radical pair, by neutralizing the negative charge on QA- and eliminating the repulsive interaction between Pheo- and QA- and/or by modifying the general dielectric properties of the protein region, surrounding these cofactors. Moreover, a close attractive interaction between Pheo-, QA-, and Cu2+ can be proposed. Our results are in good agreement with very recent EPR results indicating an additional effect of Cu2+ on the acceptor side [Jegerschöld et al. (1995) Biochemistry 34, 12747-12758].

Observation of the reduction and reoxidation of the primary electron acceptor in photosystem I.

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.

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680(+)Pheo(-), was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.

Observation of pheophytin reduction in photosystem two reaction centers using femtosecond transient absorption spectroscopy.

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.

Kinetic and Energetic Model for the Primary Processes in Photosystem II

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.