Toby D. Flint

PHOTOSYSTEM II: MAPPING THE LOCATIONS OF THE OXYGEN EVOLUTION-ENHANCING SUBUNITS BY ELECTRON MICROSCOPY

Abstract Sequential removal of the 33-, 23-, and 16-kDa extrinsic subunits of higher plant photosystem II (PSII) by various salt-washing procedures has been monitored by gel electrophoresis and oxygen evolution activity measurements. Structural changes induced by the subunit removal were followed by electron microscopy in conjunction with image processing of two-dimensional (2-D) crystals of PSII. In addition to the previously characterised 2-D crystals of native PSII and Tris-washed PSII, we report 2-D crystals of 0.25 m NaCl-washed (a= 18.2nm, b = 20.3nm with γ = 89 °) and 1 m NaCl-washed PSII (a= 18.2nm, b = 20.4nm with γ = 89 °). The three extrinsic subunits were mapped to three separate locations on the lumenal side of the complex. The structural information derived from these studies is discussed in the light of recent crosslinking and other biochemical investigations allowing a further exploration of the relationships between PSII architecture and photosynthetic oxygen evolution.

Projection structure of photosystem II in vivo determined by Cryo-electron crystallography

Abstract Photosystem II (PSII) is a protein-pigment complex situated in the thylakoid membranes of plants and cyanobacteria where it catalyses the conversion of light into chemical energy. This energy is used to extract electrons from water, during which process oxygen is evolved. Owing to its extreme fragility and the large number of polypeptides (>20) it is composed of, the complex has so far proven recalcitrant to high-resolution structural studies. Cryo-electron crystallography of 2-D crystals (a = 15.4nm, b = 23.1nm, γ = 97.2°, p1) comprising in situ PSII revealed the first projection structure of the native complex. The unit cell contain one monomeric complex in which three central domains straddle an elongated intramolecular cavity. In conjunction with earlier data, these central domains were assigned to the reaction centre core subunits of PSII consisting of CP43, CP47, the reaction centre heterodimer D1/D2 and cytochrome b-559. The data are discussed in view of the evolution of reaction centres from anoxygenic to oxygenic photosynthesis.

Rebinding of the extrinsic proteins of Photosystem II studied by electron microscopy and single particle alignment: an assessment with small two-dimensional ordered arrays of Photosystem II

Abstract Structural analysis of weakly ordered two-dimensional (2-D) ordered arrays of Photosystem II (a photosynthetic membrane protein complex) has been carried out using single-particle alignment and averaging (SPA). Although this averaging technique is normally applied to randomly oriented solubilised particles, we have found that it is also highly effective for the analysis of membrane-located 2-D ordered arrays and that the arrays can be used to check the effectiveness of the rotational alignment routines. Successful rotational alignment varied in the range 75 to 66% and 26 to 19%, depending on the stain used and hence the signal-to-noise ratio, and the data were used to address questions of space group assignments for Photosystem II 2-D ordered arrays. We thus hoped to settle the controversy as to the oligomeric form of PS II in native thylakoids, i.e., the monomeric vs. dimeric model. The methods were applied to the study of the removal and rebinding of extrinsic subunits in Photosystem II. Information on the location of the subunits in this large membrane protein complex is presented here.