The evidence that Cyt b-559 can act as an electron donor or protector to PSII reaction center

Abstract

Although Cyt b-559 is known to be closely associated with the reaction center of PSII, the property and function of Cyt b-559 are still puzzled. In this work, we have investigated the spectra characteristics and stability of purified Cyt b-559. First of all, we devised a rapid and resultful procedure of purification of Cyt b-559 from spinach and rice with using more effective chromatography media DEAE-Sephacel. From analyses of protein constituent with HPLC, it was confirmed that the Cyt b-559 has two subunits, whose molecular weights were verified as 9.4 kD and 4.5 kD, respectively. The pigment composition of the purified Cyt b-559 was analyzed by HPLC. The result revealed that the Cyt b-559 contains Chl a but no carotenoid. This was confirmed by the results of absorption spectra and resonance Raman spectrum of the purified Cyt b-559. By measurement of 77K fluorescence spectra, it was shown that the purified Cyt b-559 has two emission peaks at 563 nm and 666 nm. The results firstly proved that the Cyt b-559 emits fluorescence and can transfer excited electrons to Chl a which binds to the Cyt b-559. The above properties indicated that Cyt b-559 might be act as an electron donor to reaction center chlorophylls. From the results of photodamage of the purified Cyt b-559, it was demonstrated that the contents of His residues in Cyt b-559 were not changed. Furthermore, chemical modification with DEPC, could not modify any His residues in the Cyt b-559. These results indicated that two His residues belonging to Cyt b-559 were stable during the beginning of photoinhibition of PSII reaction center. This stability makes for exert the protective function of Cyt b-559. It suggested that under normal conditions Cyt b-559 compete with other electron donor to PSII reaction center and functions as a fuse. Under stress condition, Cyt b-559 prevents PSII reaction center from photodamage and functions as a "molecular switch" between the two forms of Cyt b-559.