Characterization of the primary processes in the photosystem II reaction center by ultrafast laser spectroscopy.

Abstract

Ultrafast transient absorption and fluorescence spectroscopy has been used to investigate the primary energy transfer and charge separation events in the Photosystem II reaction center. Transient absorption measurements, with excitation at 400 nm, on D1-D2-cyt b$\sb{559}$ reaction center complexes show that multiple excitation of reaction centers produces additional fast decay components not observed at low excitation energy. In the regime where the observed signals are linear with respect to excitation, a 20 $\pm$ 2 ps rise of the pheophytin anion absorption, bleach of the pheophytin Q$\rm\sb{x}$ absorption, and appearance of the chlorophyll cation absorption are observed. The kinetic signals are independent of the method of D1-D2-cyt b$\sb{559}$ preparation. Time-resolved fluorescence measurements of D1-D2-cyt b$\sb{559}$ reaction center complexes were made using an optical signal analyzer based on a streak camera synchronized to a femtosecond laser system. The instrumental response of this analyzer was 4-4.5 ps fwhm. Fluorescence decays were obtained throughout the Q$\rm\sb{y}$ band of PSII. These experiments show that an effective charge separation from a quasi-equilibrium of chromophore excited states is either quite fast ($<$1.25 ps) or relatively slow ($>$20 ps). Transient absorption difference measurements using selective excitation within the Q$\rm\sb{y}$ band on Tris-RCC and CP47-D1-D2-cyt b$\sb{559}$ show the existence of a red-absorbing chromophore which is coupled to the redox active pigments via a ca. 10-20 ps energy transfer process. Transient absorption measurements on D1-D2-cyt b$\sb{559}$ show that the effective charge separation rate varies as a function of excitation wavelength. The effective charge separation time constants are found to be ca. 20 ps upon blue excitation and $<$3 ps upon red excitation of the primary donor in D1-D2-cyt b$\sb{559}$. These results are supported by sodium dithionite reduction experiments, which block the charge separation reaction by pre-reducing the primary acceptor and hence allowing for only energy transfer to occur.