| The hydrophobic patch of plastocyanin (PC) from the divergent cyanobacterium Prochlorothrix hollandica possesses three atypical amino acids, Tyr12, Pro14, and Met33 instead of the highly conserved Gly, Leu, and Asn respectively. The research presented here was aimed to study protein-protein interactions of the diverse P. hollandica PC with its redox partners Photosystem I (PSI) and cytochrome f (cyt f). The unique Tyr12 of P. hollandica PC, which protrudes out of the hydrophobic patch disrupting its flat conformation, was shown to be nonessential for PC-PSI interactions as well as for the formation of a heterospecific complex between P. hollandica PC and Phormidium laminosum cytf. Moreover, reverting the unusual Pro14 and Met33 to their conserved counterparts resulted in faster electron transfer to PSI. Mutating Pro53 in order to disrupt a short alpha-helix predicted to comprise the contact area with PSI, yielded more efficient interactions, although, the ability of PC to form the complex with PSI was lost. Therefore, this site was confirmed to be indeed involved in the complex interface fixing the partners in a conformation that is not optimal for the electron transfer. Mutating the conserved Arg86, crucial for PC-PSI and PC-cytf interactions in other cyanobacteria, also increased the electron transfer rate demonstrating a different role of Arg86 in Prochlorothrix where it seems to be nonessential for PSI reduction. Overall, it has been concluded that PC-PSI interactions in Prochlorothrix are not optimized, suggesting that this organism might be using a divergent PC that appeared before evolution selected the conserved residues that optimized its reactivity. Another part of this work was devoted to the PC-cyt f interactions. Preliminary results of heterocomplex formation with Phormidium cytf illustrated that changing the Prochlorothrix PC hydrophobic pole conformation, mimicking the one of Phormidium PC, did not result in any significant changes. The binding affinity of both WT and double Y12G/P14L mutant PCs was one order of magnitude higher that that for physiological Phormidium complex. Results presented here for both physiological PC-PSI and heterospecific PC-cytf complexes have led to the conclusion that the complementarity of the surface conformation of reaction partners is not very important in transient protein-protein interactions, the latter being predominantly guided by hydrophobic forces. The studies of the physiological Prochlorothrix PC-cytf still have to be carried out. For that purpose, the sequence of Prochlorothrix cytf was determined, its structure modeled, and the protein has been expressed in sufficient amounts. |
