UBC Theses and Dissertations
Structure-based functional studies of the rhodobacter sphaeroides reaction centre H protein Tehrani, Ali
The purple bacterial photosynthetic reaction centre (RC) contains three proteins called RC H, RC M and RC L. The RC H protein comprises three cellular domains: an 11 amino acid N-terminal sequence on the periplasmic side of the inner membrane; a single transmembrane ot-helix; a large C-terminal, globular cytoplasmic domain. The roles of these domains in Rhodobacter sphaeroides RC function and assembly was investigated, using a mutagenesis approach that included domain swapping with Blastochloris viridis RC H segments, periplasmic domain deletion and site-directed amino acid changes. Additionally, possible interactions between photosynthetic RC proteins that are thought to protect these membrane proteins from proteolytic digestion in RC complex assembly were evaluated by use of translationally in-frame (non-polar) RC gene-specific deletions. The RC H, RC M and RC L proteins were expressed from plasmids, either alone or in concert with one or both of the others, in a strain of R. sphaeroides that contained chromosomal deletions of all three RC genes. The RC H periplasmic domain was shown to be involved in the accumulation of the RC H protein in the cell membrane, while the transmembrane domain has an additional role in RC complex assembly, perhaps through interactions with RC M. The cytoplasmic domain functions in RC catalytic activity and complex assembly. The RC H cytoplasmic domain surface residues His-126 and His-128 jointly mediate proton transfer into the RC by acting as proton donors at the entrance of the proton transfer pathway. There is a correlation between the amounts of membrane-associated RC H and RC L, whereas RC M is found in the cell membrane independently of RC H and RC L. Furthermore, substantial amounts of RC M and RC L are found in the soluble fraction of cells only when RC H is present in the membrane. These data were used to propose models of RC catalytic mechanisms, and RC assembly in which the RC M protein accumulates in the cell membrane regardless of the presence of the RC H and RC L proteins, and the RC M protein is a nucleus for addition of RC L followed by RC H in assembly of the RC holocomplex.
Item Citations and Data