The mechanism and kinetics of electron transfer in isolated D1͞ D2-cyt b559 photosystem (PS) II reaction centers (RCs) and in intact PSII cores have been studied by femtosecond transient absorption and kinetic compartment modeling. For intact PSII, a component of Ϸ1.5 ps reflects the dominant energy-trapping kinetics from the antenna by the RC. A 5.5-ps component reflects the apparent lifetime of primary charge separation, which is faster by a factor of 8 -12 than assumed so far. The 35-ps component represents the apparent lifetime of formation of a secondary radical pair, and the Ϸ200-ps component represents the electron transfer to the Q A acceptor. In isolated RCs, the apparent lifetimes of primary and secondary charge separation are Ϸ3 and 11 ps, respectively. It is shown (i) that pheophytin is reduced in the first step, and (ii) that the rate constants of electron transfer in the RC are identical for PSII cores and for isolated RCs. We interpret the first electron transfer step as electron donation from the primary electron donor Chl acc D1. Thus, this mechanism, suggested earlier for isolated RCs at cryogenic temperatures, is also operative in intact PSII cores and in isolated RCs at ambient temperature. The effective rate constant of primary electron transfer from the equilibrated RC* excited state is 170 -180 ns ؊1 , and the rate constant of secondary electron transfer is 120 -130 ns ؊1 .charge separation ͉ photosynthesis ͉ ultrafast spectroscopy ͉ D1͞D2-cytb559 ͉ femtosecond absorption P hotosystem (PS) II cores, whose structure has recently been determined to a resolution of 3.5-3.2 Å (1-3), consist of the antenna polypeptides CP43 and CP47, which carry 13 and 16 chlorophyll (Chl) a molecules, respectively. They contain furthermore the D1͞D2-cyt b559 reaction center (RC) polypeptides, which bind the pigments of the electron transfer chain [four Chls, two pheophytins (Pheo), and two quinones] and two additional antenna Chls (the so-called Chl z D1 and Chl z D2 molecules). The isolated RC (D1-D2-cyt b559 ) lacks the quinone acceptors and is thus only able to create a short-lived radical pair (RP) (see review in ref. 4).There exists presently no agreement on the mechanism of the primary events of energy and electron transfer in the isolated RC complex (see refs. 4-6 for recent reviews). Early studies suggested an apparent Ϸ3-ps charge separation lifetime in the RC at room temperature (7,8) in agreement with later studies (9, 10). Andrizhiyevskaya et al. (11) recently also proposed a model with an Ϸ3-ps charge separation. A somewhat slower charge separation of Ϸ8 ps has been reported by Wasielewski and coworkers (12), whereas more recent data from the same group were interpreted in terms of a 2-to 5-ps charge separation time (13). Substantially shorter charge separation times of 1 ps (14) and 0.4 ps (at 240 K) have been reported by Groot et al. (15). At the other extreme, a 1 order of magnitude longer charge separation time of Ϸ21 ps has been suggested by Klug and coworkers (16,17). Probably the largest ...

The induction and relaxation of non-photochemical quenching (NPQ) under steady-state conditions, i.e. during up to 90min of illumination at saturating light intensities, was studied in Arabidopsis thaliana. Besides the well-characterized fast qE and the very slow qI component of NPQ, the analysis of the NPQ dynamics identified a zeaxanthin (Zx) dependent component which we term qZ. The formation (rise time 10-15min) and relaxation (lifetime 10-15min) of qZ correlated with the synthesis and epoxidation of Zx, respectively. Comparative analysis of different NPQ mutants from Arabidopsis showed that qZ was clearly not related to qE, qT or qI and thus represents a separate, Zx-dependent NPQ component.

The energy transfer and charge separation kinetics in core Photosystem I (PSI) particles of Chlamydomonas reinhardtii has been studied using ultrafast transient absorption in the femtosecond-to-nanosecond time range. Although the energy transfer processes in the antenna are found to be generally in good agreement with previous interpretations, we present evidence that the interpretation of the energy trapping and electron transfer processes in terms of both kinetics and mechanisms has to be revised substantially as compared to current interpretations in the literature. We resolved for the first time i), the transient difference spectrum for the excited reaction center state, and ii), the formation and decay of the primary radical pair and its intermediate spectrum directly from measurements on open PSI reaction centers. It is shown that the dominant energy trapping lifetime due to charge separation is only 6-9 ps, i.e., by a factor of 3 shorter than assumed so far. The spectrum of the first radical pair shows the expected strong bleaching band at 680 nm which decays again in the next electron transfer step. We show furthermore that the early electron transfer processes up to approximately 100 ps are more complex than assumed so far. Several possibilities are discussed for the intermediate redox states and their sequence which involve oxidation of P700 in the first electron transfer step, as assumed so far, or only in the second electron transfer step, which would represent a fundamental change from the presently assumed mechanism. To explain the data we favor the inclusion of an additional redox state in the electron transfer scheme. Thus we distinguish three different redox intermediates on the timescale up to 100 ps. At this level no final conclusion as to the exact mechanism and the nature of the intermediates can be drawn, however. From comparison of our data with fluorescence kinetics in the literature we also propose a reversible first charge separation step which has been excluded so far for open PSI reaction centers. For the first time an ultrafast 150-fs equilibration process, occurring among exciton states in the reaction center proper, upon direct excitation of the reaction center at 700 nm, has been resolved. Taken together the data call for a fundamental revision of the present understanding of the energy trapping and early electron transfer kinetics in the PSI reaction center. Due to the fact that it shows the fastest trapping time observed so far of any intact PSI particle, the PSI core of C. reinhardtii seems to be best suited to further characterize the electron transfer steps and mechanisms in the reaction center of PSI.

Photosystem I (PSI) is a large pigment-protein complex that unites a reaction center (RC) at the core with ∼100 core antenna chlorophylls surrounding it. The RC is composed of two cofactor branches related by a pseudo-C2 symmetry axis. The ultimate electron donor, P 700 (a pair of chlorophylls), and the tertiary acceptor, F X (a Fe 4 S 4 cluster), are both located on this axis, while each of the two branches is made up of a pair of chlorophylls (ec2 and ec3) and a phylloquinone (PhQ). Based on the observed biphasic reduction of F X , it has been suggested that both branches in PSI are competent for electron transfer (ET), but the nature and rate of the initial electron transfer steps have not been established. We report an ultrafast transient absorption study of Chlamydomonas reinhardtii mutants in which specific amino acids donating H-bonds to the 13 1 -keto oxygen of either ec3 A (PsaA-Tyr696) or ec3 B (PsaBTyr676) are converted to Phe, thus breaking the H-bond to a specific ec3 cofactor. We find that the rate of primary charge separation (CS) is lowered in both mutants, providing direct evidence that the primary ET event can be initiated independently in each branch. Furthermore, the data provide further support for the previously published model in which the initial CS event occurs within an ec2/ec3 pair, generating a primary ec2 þ ec3 − radical pair, followed by rapid reduction by P 700 in the second ET step. A unique kinetic modeling approach allows estimation of the individual ET rates within the two cofactor branches.Chlamydomonas | electron transfer directionality | femtosecond absorption | photosystem I | ultrafast spectroscopy I n oxygenic photosynthesis, the primary reactions of light utilization are driven by two multisubunit, pigment-protein complexes-photosystem II (PSII) and photosystem I (PSI) (1-3). The structures of PSI from the cyanobacterium Thermosynechococcus elongatus (4) and from the plant Pisum sativum (5) have been resolved to 2.5 Å and 3.4 Å, respectively. The PSI core complex consists of an extensive antenna (ANT) system of ∼100 densely packed Chls and a relatively isolated group of redox (reduction-oxidation reaction) active cofactors at the center composing the reaction center (RC). As in all other RCs, the cofactors in the RC of PSI form two quasi-symmetric branches (Fig. 1), diverging from a Chl a 0 ∕Chl a pair (ec1 A ∕ec1 B ) traditionally called P 700 (4). In each branch is a pair of Chl a molecules (ec2 A ∕ec3 A or ec2 B ∕ec3 B ) and a phylloquinone (PhQ A or PhQ B ). Finally, the branches join again at the F X iron-sulfur (FeS) cluster.The symmetry of the cofactor branches is key to discussion of the directionality of electron transfer (ET) in PSI. Originally, it had been assumed that, analogous to the type II RCs, ET in PSI proceeded along only one of the cofactor branches, and that the other branch was not used. This idea would have been supported by the structural asymmetry of P 700 , if P 700 were the primary electron donor in PSI. However, evidence was provided that muta...

The energy transfer kinetics from carotenoids to chlorophylls and among chlorophylls has been measured by femtosecond transient absorption kinetics in a monomeric unit of the major light-harvesting complex (LHCII) from higher plants. The samples were reconstituted complexes with different carotenoid contents. The kinetics was measured both in the carotenoid absorption region and in the chlorophyll Q(y) region using two different excitation wavelengths suitable for selective excitation of the carotenoids. Analysis of the data shows that the overwhelming part of the energy transfer from the carotenoids occurs directly from the initially excited S(2) state of the carotenoids. Only a small part (<20%) may possibly take an S(1) pathway. All the S(2) energy transfer from carotenoids to chlorophylls occurs with time constants <100 fs. We have been able to differentiate among the three carotenoids, two luteins and neoxanthin, which have transfer times of approximately 50 and 75 fs for the two luteins, and approximately 90 fs for neoxanthin. About 50% of the energy absorbed by carotenoids is initially transferred directly to chlorophyll b (Chl b), while the rest is transferred to Chl a. Neoxanthin almost exclusively transfers to Chl b. Due to various complex effects discussed in the paper, such as a specific coupling of Chl b and Chl a excited states, the percentage of direct Chl b transfer thus is somewhat lower than estimated by us previously for LHCII from Arabidopsis thaliana. (Connelly, J. P., M. G. Müller, R. Bassi, R. Croce, and A. R. Holzwarth. 1997. Biochemistry. 36:281). We can distinguish three different Chls b receiving energy directly from carotenoids. We propose as a new mechanism that the carotenoid-to-Chl b transfer occurs to a large part via the B(x) state of Chl b and to the Q(x) state, while the transfer to Chl a occurs only via the Q(x) state. We find no compelling evidence in favor of a substantial S(1) transfer path of the carotenoids, although some transfer via the S(1) state of neoxanthin can not be entirely excluded. The S(1) lifetimes of the two luteins were determined to be 15 ps and 3.9 ps. A detailed quantitative analysis and kinetic model of the processes described here will be presented in a separate paper.