On the basis of rational design principles, template-assisted four-helix-bundle proteins that include two histidines for coordinative binding of a heme were synthesized. Spectroscopic and thermodynamic characterization of the proteins in solution reveals the expected bis-histidine coordinated heme configuration. The proteins possess different binding domains on the top surfaces of the bundles to allow for electrostatic, covalent, and hydrophobic binding to metal electrodes. Electrostatic immobilization was achieved for proteins with lysine-rich binding domains (MOP-P) that adsorb to electrodes covered by self-assembled monolayers of mercaptopropionic acid, whereas cysteamine-based monolayers were employed for covalent attachment of proteins with cysteine. Immobilized residues in the binding domain (MOP-C) proteins were studied by surface-enhanced resonance Roman (SERR) spectroscopy and electrochemical methods. For all proteins, immobilization causes a decrease in protein stability and a loosening of the helix packing, as reflected by a partial dissociation of a histidine ligand in the ferrous state and very low redox potentials. For the covalently attached MOP-C, the overall interfacial redox process involves the coupling of electron transfer and heme ligand dissociation, which was analyzed by time-resolved SERR spectroscopy. Electron transfer was found to be significantly slower for the mono-histidine-coordinated than for the bis-histidine-coordinated heme. For the latter, the formal heterogeneous electron-transfer rate constant of 13 s(-1) is similar to those reported for natural heme proteins with comparable electron-transfer distances, which indicates that covalently bound synthetic heme proteins provide efficient electronic communication with a metal electrode as a prerequisite for potential biotechnological applications.
|Publication status||Published - 2005|