Photo-oxidation of tyrosine in a bio-engineered bacterioferritin 'reaction centre'

Abstract

The photosynthetic reaction centre (RC) is central to conversion of solar energy into chemical energy. In this thesis, in order to introduce the redox-active cofactors similar to that of the Photosystem II RC, a non-photosynthetic protein scaffold was used as an in vitro model. The protein tasked for this purpose was the heme containing bacterioferritin (BFR) protein found in E. coli. The BFR protein naturally expresses as a homodimer based on a 4-helix bundle monomer. Desirable properties included: (i) a promiscuos di-nuclear metal binding site which provides ligands for class II metals such as Mn (ii) a hydrophobic pocket at the dimer interface which can bind a photosensitive porphyrin, in this case a chlorin (Ce6), and (iii) presence of tyrosine residues proximal to the bound cofactors, which can be utilised as efficient electron-tunnelling intermediates. The work in this thesis extends earlier work in the group by refining and improving the BFR system. Several mutants were made and an improved protein expression system was developed. For these samples experiments demonstrated ligation of weakly coupled equivalent Mn2II,II at the di-nuclear binding site of apo-BFR, and binding of the photo-active pigment ZnCe6 in hydrophobic pocket of the protein. Light-induced electron transfer from proximal tyrosine residue(s) to the photo-oxidised ZnCe6+, in the modified BFR reconsitituted with both ZnCe6 and MnII is presented. Three site-specific tyrosine mutants (Y25F, Y58F and Y45F) were made to localise the redox-active tyrosine in this engineered system. The results indicate that: (i) presence of bound MnII is necessary to observe tyrosine oxidation in all BFR variants, (ii) Y45 (within van Der Waals network of ZnCe6) is singly the most important tyrosine as the immediate electron donor to the oxidised ZnCe6+, and (iii) Y25 and Y58 are both redox-active in this system, but appear to be interchangebale. A high-resolution (~1.5 Ã…) crystal structure of the three tyrosine mutants was obtained and these structures showed there to be no mutation-induced effects on the overall 3-D structure of the protein. Minimal effects observed in the Y45F mutant are reported. The molecular design of a "second generation" of the BFR series is also presented where the symmetry of the BFR homodimer is broken to generate a BFR heterodimer to contain genetically distinct electron donor and acceptor subunits. The BFR heterodimer was made by introducing a small (20 aa) peptide linker between the two subunits. The ultimate goal will be to demonstrate light-induced directional electron flow in the BFR heterodimer. Show more