Isolation of the extrinsic proteins from the oxygen-evolving complex of higher plants

Jaroslava Kohoutova¹, Olga Shmidt¹ and Ivana Kuta Smatanova^1,2

¹Institute of Physical Biology, University of South Bohemia in České Budějovice, 373 33 Nové Hrady, Czech Republic

²Institute of Systems Biology and Ecology of the Academy of Science of the Czech Republic, v.v.i., 373 33 Nové Hrady, Czech Republic

In oxygenic photosynthesis sunlight is converted into chemical energy and the oxygen (essential for life on earth) is released to the atmosphere. Water splitting, giving the rise to molecular oxygen, is performed on a cluster of four Mn²⁺ ions located on the lumenal side of photosystem II (PSII). For the optimal activity of this oxygen-evolving complex are required Ca²⁺ and Cl^- ions, which is modulated in higher plants by the presence of three extrinsic proteins named PsbO, PsbP and PsbQ and create the correct ionic environment during water oxidation. These proteins are located at the lumenal surface of membrane [1]. Additionally, the protein PsbR has been described to play important role in water oxidation in plant PSII [2,3].

The isolation of the protein complex consisting of proteins PsbO, PsbP, PsbQ and the extrinsic protein PsbR from Pisum sativum L. for other structural analysis was optimized. Western blotting was used for detection of extrinsic proteins and preliminary crystallization experiments were performed. It was shown that complex PsbO, PsbP, PsbQ is stable in the Tris buffer, pH 8.6 while for protein PsbR is sufficient Tris buffer pH 7.00 containing the detergent octyl-thioglucopyranoside.

[1] A. Seidler, Biochem.Biophys. Acta 1277 (1996) 35–60

[2] U. Ljungberg, H.-E. Ĺkerlund, B.Andersson, Eur. J. Biochem. 158 (1986) 477–482

[3] M. Suorsa, S. Sirpio, Y.Allahverdiyeva, V. Paakkarinen, F.Mamedov, S.Styring, E. Aro, J. Biol. Chem. 281 (2006) 145–150

This work is supported by grants COST Xtall LD11011, LC06010, MSM6007665808 of the Ministry of Education of Czech Republic, by grant AV0Z60870520 of AS CR and work of O.S. is supported by grant GAJU 170/2010/P.