Photosystem II PsbO protein from higher plants

Jiří Heller¹, Daryna Kulik¹, Ondřej Šedo², Zbyněk Zdráhal², Ivana Kutá Smatanová^1,3 and Jaroslava Kohoutová¹

¹University of South Bohemia in České Budějovice, Faculty of Science, Branišovská 31, 37005 České Budějovice
²Core Facility – Proteomics, CEITEC, Masaryk University, Kamenice 5,625 00 Brno
³Academy of Sciences of the Czech Republic, Inst. of Nanobiology and Structural Biology GCRC, Zámek 136, 373 33 Nové Hrady

Photosystem II (PSII) is a huge complex of proteins in thylakoid membranes of algae, higher plants, and cyanobacteria that conducts light-driven water oxidation and produces molecular oxygen, electrons and protons. The splitting of water and release of oxygen appear in the catalytic centre of PSII – the oxygen-evolving centre (OEC) that contains manganese-calcium cluster (4:1 Mn:Ca) situated near to the luminal surface of the transmembrane domain and hemmed by intrinsic and extrinsic components in thylakoid membranes. PsbO (33kDa), PsbP (23kDa), PsbQ (17 kDa), PsbR (10 kDa) are extrinsic proteins attached to the luminal side of PSII in higher plants, which keep stability of water oxidation site and right ionic environment during oxidation of water. The aim of our project is to receive recombinant PsbO proteins from Spinacia oleracea and Pisum sativum. The isolation of mRNA from leaves and convertion to cDNA were our first attempts to obtain the psbO gene. The conditions of polymerase chain reaction (PCR) were adjusted and DNA fragments encoding the psbO gene were acquired. Vector pBluescript II SK(+) was utilized as a cloning vector and pET-28b(+) vector was employed for overexpression of recombinant PsbO/HisPsbO proteins. Purification, crystallization and structure designation of the recombinant proteins PsbO/HisPsbO of the oxygen-evolving complex from Pisum sativum and Spinacia oleracea are under construction.