Excitation dynamics in eukaryotic PS I from Chlamydomonas reinhardtii CC 2696 at 10 K. Direct detection of the reaction center exciton states

Excitation energy transfer in PS I particles from the green alga Chlamydomonas reinhardtii CC 2696 was studied at 10 K by femtosecond transient absorption spectroscopy. Five-nm wide excitation pulses at 670, 680, 695, and 700 nm were applied to selectively excite different spectral forms contributing to the wide Q_Y transition band of chlorophyll a. Absorbance changes between 630 and 770 nm, up to 100 ps after excitation, were collected with a time step of 54 fs during the first 5 ps. Excitation at 700 nm leads to a structured initial absorbance difference spectra with four positive bands clearly resolved at 634, 645, 652, and 661 nm, and four negative bands at 667, 675,684, and 695 nm. These spectra are interpreted in terms of excitonic coupling between the six electron-transfer chlorophyll a molecules: a special pair, two accessory and two A₀ chlorophylls. The negative bands were ascribed to photobleaching of the four one-exciton states in line with theoretical predictions (Beddard, G.S. J. Phys. Chem. B. 1998, 102, 10 966), and the positive ones to excited-state absorption. The significance of the broad absorbance changes is proposed to be the introduction of spectral overlap between the reaction center and different spectral forms of the antenna chlorophylls that is expected to increase the efficiency of energy flow to the reaction center. Excitation at different wavelengths shows indeed that trapping can occur from different spectral pools of chlorophylls with similar efficiency (trapping time 29-44 ps). Following the excitation at 670 and 680 nm, trapping was shown to occur from the same pool as at room temperature centered at 682-685 nm, containing apparently only a minority of antenna molecules located close to the reaction center. The trapping time was found to be only slightly longer compared to that at room temperature (20-23 ps at RT). At 10 K, a significant amount of chlorophylls cannot exchange excitation energy with their neighbors. Our results are consistent with previous reports that at cryogenic temperatures, charge separation is possible in ∼50% of PS I particles and that excitation quenching by the oxidized and reduced primary donor is equally effective. As was observed at room temperature, there is no indication of red chlorophylls absorbing above 700 nm. This lack of red chlorophylls makes it possible to directly excite reaction center chlorophylls and study interaction between them in wild type and, in future, mutant PS I from Chlamydomonas.