Photosystem ¢ò Reaction Center Of Cytochrome B559 Structure And Function

Life on earth is sustained by photosynthesis. Through about two billion yearsevo1ution, higher plants have evolved several mechanisms to prevent photoinhibitionand photodamage from high irradiance stress. Although Cyt b-559 is known to bec1osely associated with the reaction center of PSIl, the property and function of Cyt b-559 are Still puzzled. ln this work, we have investigated the characteristics and thephysiological functions of Cyt b-559. All the results were summarized as followings fl. According to the principle of purification of PSIl reaction center, we devised arapid and resultful procedure of purification of Cyt b-559 from spinach and rice,By using more effective chromatography media DEAE~Sephacel, we obtainedhighly purified Cyt b-559. Purified Cyt b-559 from these species have similarmobility during gel electrophoresis under native conditions. From analyses ofprotein constituent with HPLC, it was determined that the Cyt b-559 has twosubunits. From the results of Tricine-SDS-PAGE, which has superior resolution ofsmalI proteins, molecuIar weight of a and 0 subunits of the Cyt b-559 were verifiedas 9.4 kD and 4.5 kD, respectively.2. The pigment composition of the purified Cyt b-559 was analyzed by HPLC. Theresu1t revealed that the Cyt b-559 contains Chl a but no carotenoid. This wasconfirmed by the results of absorption spectra and resonance Raman spectrum ofthe Cyt b-559, By using IEF we anaIyzed the pI of the Cyt b-559 holoprotein andits two subunit. lt was found that the pIs of two subunits are different and they alsodiffer to the pI of Dl or D2. We suggested that opposite electric polarity betweenCyt b-559 and Dl under physiological condition may favor construction of PSII.3. By measurement of low temperature (77K) fluorescence spectra, it was shown thatthe purified Cyt b-559 has two emission peaks at 563 nm and 666 nm. The resultsfirstly proved that the Cyt b-559 emits fluorescence and can transfer excitede1ectrons to Chl a which binds to the Cyt b-559. By using uItraviolet fluorescencespectra for the first time, we demonstrated that Trp residues locate in hydrophobictransmembrane domain of the Cyt b-559. It was supposed that these amino acidsaffect the function of Cyt b-559.4. It was observed that the Cyt b-559 have MCD signals in 540-580 nm region and400-440 nm region. The spectrum and intensity of MCD of the purified Cyt b-559are identical to PSII reaCtion center in these regions, it was inferred that the Cyt h-559 is one of contributors fOr MCD of PSII reaction center, The results of FTIRmeasurement proved that His residues are ligands of heme of the Cy''t b-559 and a-helixes are central kind of secondary structures in Cyt b-559. ln addition, we havecompared the Iipid contents of the purified Cyt b-559 and PSII reaction center. Theabove properties of Cyt b-559 purified from different higher plants are very similar.These results indicated that Cyt b-559 is very conservative during evolution.5. Our results confirmed that the primary donor (P680) of PSII reaction center wasalways finally photodamaged whether under acceptor-side photoinhibition or underdonorside photoinhibition. We found that during the initial stage ofphotoinhibition, the absorption of Cyt b-559 was increased under acceptor-sidephotoinhibition but decreased under donor-side photoinhibition. These phenomenashowed that Cyt b-559 is very sensitive to photoinhibition and may protect PSllreaction center on the beginning of photoinhibition.6. From the results of photodamage of the purified Cyt b-559 under similarphotoinhibition condition, it was demonstrated that the contents of His residues inCyt b-559 were not changed. Furthermore, DEPC, which can modify some Hisresidues in PSII reaction center, could not modify any His residues in the Cyt b-559.These results indic8ted that two His residues belonging to Cyt b-559 were stableduring the beginning of photoinhibition of PSII reaction center. T...