Metalloproteins play important roles in biology, as they account for nearly half of all proteins in nature, spanning a remarkable functional range, from fundamental chemical reactions, such as electron transfer, dioxygen transport and activation, hydrolysis, group transfer and rearrangements, to signaling processes governing gene regulation and expression. The same metal cofactor is able to serve different roles depending on the protein architecture, as the physical and chemical properties of a selected metal depends on the protein environment. In fact, a plethora of mixed interactions occur between the metal cofactor and the protein matrix, fine-tuning the intrinsic reactivity of the metal. A prominent illustration of how the metal center reactivity is modulated by the surrounding protein environment is provided by heme-proteins, that are a family of important macromolecules ubiquitous in biological systems characterized by a common prosthetic group (the “heme” group), which perform a wide range of biological functions, and participate in a wide range of essential biological processes: myoglobin and hemoglobin reversibly bind dioxygen, cytochromes support electron-transfer processes, cytochrome P450 activates dioxygen to form water and an oxygen atom, which is inserted into a substrate. A powerful approach to deeply understand the factors that specify the functions of natural heme-proteins is the engineering of peptide-based models that mimic protein active sites in simplified scaffolds. To date, several artificial heme-proteins have been engineered, providing provide further insights on -activity relationships. In this respect, the Artificial Metalloenzyme group, where this project has been carried out, developed a class of artificial heme-mimetics, named Mimochromes. These models are composed of two peptide chains covalently linked to the heme group via amide bonds between the porphyrin propionates and the lysine side-chains. Early compounds of this class were made up of two identical nona-peptide chains, each containing a histidine residue which acts as axial ligand to the heme central ion (iron or cobalt), in a low-spin bis-His-ligated state. The peptide chains were in an alpha-helical conformation in both the apo and metal containing species, and these molecules adopt a stable and well defined conformation, being able of supporting electron transfer processes. More recently, many efforts have been made in order to design new compounds (Mimochrome VI and its analogues) that bind iron in five-coordinate complexes, with a vacant coordination site available for catalyzing oxidation reactions. This efforts lead to the development of new models that have peroxidase-like activity, being able of catalyzing organic substrate (such as ABTS) oxidation, by the use of hydrogen peroxide. In particular, one of this analogues, showed catalytic performance approaching that of HRP. One main drawback of these new molecules relies in the lack of detailed information of their three-dimensional structures. Attempts to solve the structure on the fully diamagnetic Co(III) complex were unsuccessful. In fact, analysis of the Nuclear Magnetic Resonance (NMR) spectrum of Co(III)-Mimochrome VI revealed the presence of different species in solution. This PhD project was aimed at overcoming the presence of multiple species in solution, developing heme-mimetics that couple high activity with a well defined structure. In this respect, two different strategies have been undertaken: * incorporation of non-coded amino acid with alpha-helix stabilizing effect; * elongation of the peptide chains, that allows the introduction of a large number of intra-chain and inter-chain interactions. The first strategy led to the development of a new Mimochrome VI analogue, named Mimochrome VI-2U1L. The second strategy resulted in the design of the Mimochrome VII molecule. For both compounds, a detalied NMR analysis was performed, and information about the three- dimensional structure of the Co(III)- complexes was achieved. For Co(III)-Mimochrome VI-2U1L, four different species were present in soultion. The four species were purified by RP-HPLC and separately analyzed by NMR. In particular, for two of these species (peak a and peak b), the solution structures of the tetradecapeptide-porphyrin moiety was solved. The tetradecapeptide adpots in solution the expected helical conformation in the Asp1 -Lys9segment, while distortion is observed at the C-terminal region (intended to fold in extended conformation). Regarding the decapeptide chain, severe overlaps make difficult the structural characterization. In order to solve the oserved ambiguity of assignment, further experiments are required. Indeed, the information obtained from the Co(III)-Mimochrome VI-2U1L molecule, will be very useful in order to analyze the structure-function relationship on Mimochorme VI and its analogues. Regarding the Mimochrome VII molecule, two species were presnt in solution. The structure of the more abundant specie was determined and good agreement was observed between the designed model and the determined NMR structure, apart from the first 4 residues at the N-terminal region. The two peptide chains adopt right-handed helical conformation from Pro6 to Leu16, while a less regular conformation is observed in the N-terminal region. As designed, the global structure is stabilized by the presence of interactions between hydrophobic side chains and deuteroporphyrin ring. These information will be used to re-design new molecules characterized by asymmetrical metal binding site, in order to introduce new functionalities. Preliminary, in this PhD thesis the design of a penta-coordinated model, Mimochrome VII asym, was carried out. This new model is characterized by a distal cavity that contains an arginine residue, mimicking Arg38 of Horseradish Peroxidase (HRP), and a non-coordinating His residue to mimic His42 of HRP. Its characterization is actually under course. Finally, part of this work was devoted to the structural characterization of active sites in paramagnetic iron(III) mimochrome complexes. In particular, the orientation of the histdine axial ligands in the Fe(III) complexes of mimochrome IV was determined, by taking advantage of a methodology previously used for natural ferriheme-proteins.