Structure and Function of the Psb27 subunit of Photosystem II

Abstract

Photosystem II is a large protein complex that catalyses the light-driven oxidation of water. The biogenesis of Photosystem II is a regulated process that involves transient interactions between Photosystem II and dedicated Photosystem II assembly proteins. One such protein, Psb27, associates preferentially with inactive Photosystem II complexes. This study was initiated to discover the structural basis of the interaction between Psb27 and Photosystem II. In this thesis, the solution NMR structure of the Psb27 protein from the cyanobacterium Synechocystis sp. PCC 6803 was solved. The Psb27 protein was found to be composed of four alpha helices arranged in a right-handed up-down-up-down topology (α1, residues 11-26; α2, 35-52; α3, 63-82; α4, 89-108). Residues 5-10 of the amino-terminus lie in a shallow cleft formed by helices 1 and 2. The position of the amino-terminal tail appears to be stabilised by hydrogen bonds between Asp14 and the backbone amides of Thr8 and Gly9. Mutation of Asp14 to Ala resulted in destabilisation of Psb27, as indicated by the apparent melting temperature of the Psb27(D14A) protein determined by circular-dichroism spectroscopy. The Psb27 protein is highly conserved amongst oxygenic-photosynthetic organisms. Thirteen highly conserved residues were identified on the surface of Psb27 (Arg54, Thr70, Asn73, Ser74, Tyr78, Ser81, Tyr82, Pro86, Pro88, Arg94, Glu98, Glu103 and Arg108). The majority of these residues are located in a group centred on helix three. This group of residues may be involved in the interaction between Psb27 and Photosystem II. All of the conserved surface exposed residues of Psb27 were subject to site-directed mutagenesis in order to test the contribution of each residue to Psb27 function in vivo. To screen for Psb27 function the following mutants were created in the genetic background of a light-sensitive ΔPsbT strain of Synechocystis sp. PCC 6803: D14A, R54E, R94E, E98R, E103R and R108E. Both the ΔPsbT:Psb27(D14A) and ΔPsbT:Psb27(R94E) strains had reduced ability to recover from photodamage. This suggests that destabilisation (D14A) or an alteration in the surface properties (R94E) of Psb27 result in loss of Psb27 function. The Psb27(R94E) mutation in the wild-type background of Synechocystis sp. PCC 6803 resulted in the assembly of active Photosystem II complexes with altered plastoquinone oxidation kinetics. This suggests that Psb27 has an influence on the composition of the active Photosystem II complex.