Studies On The Structure And Function Of Extrinsic 33 KDa Protein Of Photosystem Ⅱ

In higher plants, manganese cluster plays a crucial role in the oxygen evolution of photosystem II. Three extrinsic proteins, named 17, 23 and 33 kDa protein, binding at the lumen side of thylakoid membranes, are also necessary for efficient PSII oxygen evolution. Among them, 33 kDa protein has special significance, as it stabilizes the manganese cluster under physiological conditions. Structure and function of extrinsic 33 kDa protein were studied in this paper, and the main results includes following three parts:1. Influences of low pH and NBS modification of Trp241 upon conformational changes in 33 kDa proteinThe studies of both circular dichroism (CD) and fluorescence spectra showed that upon decreasing pH from 6.2 to 2.5, the conformation of soluble 33 kDa protein changed significantly, with an increase of random coil. The secondary structures of the protein recover upon increasing pH to neutral pH. After NBS modification at pH 2.5, the content of random coil becomes higher. Moreover, the conformational flexibility is lost in NBS-modified protein, and conformational change is pH irreversible, indicating that NBS modification blocked the reversibility of protein's conformational change. The specific binding ability of NBS-modified protein is much lower than that of low pH-treated 33 kDa protein. Furthermore, the rebinding of modified protein on PSII membranes can not restore the oxygen evolution. We suggest that it is not NBS modification of Trp241 but low pH, leading to the conformational change of 33 kDa protein from a functional to another non-functional state, and Trp241 may be a key residue for the sequential deprotonation of correlated amino acids that induces a functional conformation of 33 kDa protein.2.pH-induced conformational changes in the soluble 33 kDa proteinpH-induced changes in the conformational states of 33 kDa protein has been studied using fluorescence, far-UV CD, and pressure-induced unfolding at varying suspension pH values. Four conformational states of 33 kDa protein were identified when suspension pH was altered from 2 to 12. 33 kDa protein is completely unfolded at a suspension pH above 11, and partly unfolded below a pH of 3. The structure of 33 kDa protein appears stably folded around pH 6 and 4. The conformational state of 33 kDa protein at pH 4 seems more stable than that at pH 6. A decrease in the suspension pH to 2 resulted in significant alterations in the 33 kDa protein structure, suggesting the presence of a large number of unprotonated amino acid residues possibly used for proton transport in oxygen evolution. pH-induced structural changes of stable 33 kDa protein (pH 6 to 4) might be utilized to analyze its function as a cofactor for oxygen evolution.3.Installation of a chlorophyll a (Chl a) fluorometer and Investigation of influence of PSII donor side on Chl a fluorescence inductionA computer-controlled Chl a fluorometer was assembled and related data collecting and analysis programs were compiled. With this fluorometer, influence of PSII donor side on Chl a fluorescence induction was studied. Results showed that treatments on PSII donor side influence the maximum level and the kinetics of Chl a fluorescence, suggesting that the donor side of PSII causes influence on Chl a fluorescence induction, and manganese cluster and extrinsic proteins might effect in different ways.