| All of the charged residues in Chlamydomonas reinhardtii (C. reinhardtii) cytochrome c6 (cyt c6) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f(cyt f) were modeled using Brownian Dynamics (BD) simulations. Systematic mutation of charged residues on both PC and cyt c6 confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and Van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c6 displayed similar inhibition classes. My results indicated a difference between the two acidic clusters on PC. Mutations D44A and E43A, in the lower cluster showed greater inhibition than any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c6 that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. As there are no experimental c6 mutation data available for algae, my results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement.; The availability of seven different structures of cyt f from C. reinhardtii allowed me, using BD simulations, to model interactions between these molecules and their redox partners, PC and cyt c6 in the same species to study the effect of cyt f structure on its function. Different cyt f structures, which are very similar, produced different reaction rates in interactions with PC and cyt c6. I was able to attribute this to structural differences among these molecules, particularly to a small flexible loop between A184 and G191, on the cyt f small domain. I also showed that deletion of the cyt f small domain affected cyt c6 more than PC, due to their different binding positions on cyt f.; The availability of C. reinhardtii cytochrome b6f complex, PC, and cyt c6 structures allowed me, for the first time, to model electron transfer interactions between the extramembrane segments of this complex (luminal domains of cyt f plus Rieske subunits) and its redox partners in the same species. The Rieske protein in the both available crystal structures of the cyt b6f complex did not interfere with binding of PC or cyt c6 on cyt f. When Rieske was moved close to the cyt f in C. reinhardtii, it even enhanced the interactions. PC docked on C. reinhardtii cyt f with the same orientation in the presence or the absence of the Rieske protein, which matched well with the previously reported NMR structures of complexes between cyt f and PC. The structural studies on cyt f showed that the cyt f structure is a more important factor in the rate of complex formations than is the presence or the absence of the Rieske protein, or its position with respect to cyt f. (Abstract shortened by UMI.)... |
