Synthetic metalloproteins as electron transfer models: Design, characterization and electron transfer properties

This dissertation presents the design, characterization and electron transfer (ET) properties of two generations of de novo synthetic metalloproteins.; These peptide systems are based on the α-helical coiled-coil structural motif which is used to separate metal-based redox sites across a well-defined, yet non-covalent protein interface. The first generation metalloprotein in this study is represented by H21(30-mer), a polypeptide that forms a stable, self-assembled α-helical coiled-coil in aqueous solution. The coordination of ruthenium-based electron acceptor precursor ([Ru(trpy)(bpy)]2+) and donor ([Ru(NH₃)₅]2+) to a histidine residue on each of its two separate polypeptide chains transforms H21(30mer) into an electron transfer active metallopeptide. ([Ru(trpy)(bpy)]2+ ) precursor can be then oxidized into ([Ru(trpy)(bpy)]3+) species in a reaction with strongly oxidizing agent, and ([Ru(NH₃) ₅]2+) donor reduces these oxidized species in a thermodynamically favorable (ΔG0 = −1.11 eV) electron transfer process. Importantly, this design leads to the creation of a statistical mixture of homodimers, having only a donor or only an acceptor sites, and heterodimer, having a donor on one chain and an acceptor on another. Only the heterodimer can display intra-complex electron transfer, whose kinetics was measured by pulse radiolysis yielding a rate constant of 380 s−1 for this process occurring over a metal-to-metal distance of 24 Å. This system is the first protein-like model with ubiquitous secondary structure in which ET occurs across the noncovalent interface. Future investigation of these artificial redox proteins should produce additional insight into the mechanisms of biological ET reactions.; The second generation of ET metallopeptides studied here is represented by the K(37-mer) and E(37-mer) peptides modified with ruthenium-complexes. These polypeptides are designed such that when they are in an equimolar mixture they selectively form an ET heterodimer in a process controlled by interhelical electrostatic interactions. This improved design leads to the generation of only the ET active species, heterodimer with a donor and an acceptor on each peptide chain. Another feature of this new system is that oppositely charged amino acid residues are positioned at the solvent-exposed surface of each peptide to provide the ability to test protein-protein interactions and their recognition mechanism. ET measurement showed the occurrence of concentration dependent process only, which was assigned to intermolecular ET process. Obtained ET data was analyzed in term of Debye-Huckel theory to show that the local charges on the redox centers rather than the surface charges determine the ET properties in this system. Additionally, the electrostatic attraction between the oppositely charged peptide surfaces has shown to result in screening effect on the effective charge of the redox centers interaction.