| In nature, metalloproteins account for nearly half of all proteins and are responsible for catalyzing important biological processes, such as photosynthesis, respiration, water oxidation, molecular oxygen reduction and nitrogen fixation. Hemes are one of the most abundant and versatile cofactors in metalloproteins utilized in biology. For these reasons, scientists have sought to recapitulate the properties of heme proteins in model systems. Until now, though, little has been done to model membrane hemoproteins.;ME1, a functional membrane metalloprotein, was designed by Ghirlanda et al by engineering a bis-histidine heme binding site into a natural membrane protein, glycophorin A (GpA). A single-point mutant of ME1, G25F, modulates the redox potential of the cofactor by 150 mV. This is the largest effect observed for a single point mutant in a peptide model system. G25F was designed to test whether aromatic-porphyrin interactions can modulate the properties of the heme complex in the context of a membrane protein. Another mutant, R27F, was designed to prove the importance of orientations of aromatic residue around the heme binding pocket. R27F with apparent dissociation constant of (7.45 +/- 2.2) x 10-7 M and midpoint potential of -136 mV has similar binding affinity for ferric hemin and midpoint potential as ME1. Thus, R27F behaves like ME1, where the aromatic residues are not part of the hydrophobic core. These results prove that heme in G25F is more sensitive to aromatic stacking than previously observed in model systems or in natural membrane proteins. These results present a cautionary tale on the use of water soluble systems as models for membrane embedded hemoproteins, and point to the need for systematic, quantitative analysis of the factors affecting properties of cofactors in minimalistic membrane systems.;Recent studies on designed hemoprotein models, as well as the G25F mutant, have shown a clear inverse relationship between binding affinity and peroxidase activity. Thus, an assembly of a penta-coordinated analogue of ME1 was attempted using different chemical ligation strategies, which were used to construct ME1 heterodimer and to ligate a porphyrin covalently with a monomer peptide. This assembly of the penta-coordinate analogue of ME1 is designed to support higher levels of catalysis.;Photosystem II (PSII) has a metal based oxygen evolving complex (OEC); the structure of this metalloprotein is not completely known. A maleimide base chemical ligation, as well as a PNA based strategy, is proposed to hold three peptide components together in a DNA tetrahedron with a geometry mimicking the 3D structure of the three peptides in Photosystem II. These three peptides are known coordinate with the 4MnCa cluster that catalyzes the water oxidation reaction. |
