Artificial heme-enzymes for catalytic and diagnostic applications

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Through the study of natural proteins and model systems, it is becoming increasingly clear that different proteins are able to optimize the environment surrounding the heme-cofactor for the acquisition of specific functions. In this respect, this Ph.D. project was focused on the development of new artificial heme-protein mimetics, belonging to the Mimochrome family. Mimochromes are made up by two peptide chains, covalently linked to the deuteroporphyrin IX. Their global folding can be viewed as a sandwich, in which the peptide chains in α-helical conformation cover both planes of the heme. Originally designed to mimic the bis-His cytochrome b, the Mimochrome structure was modified to introduce a peroxidase-like activity, by creating a distal cavity on the heme. The success with the first asymmetric system, Fe(III)-Mimochrome VI, gave the opportunity to explore further modifications in order to improve the catalytic activity. Therefore, four Mimochrome VI analogues were developed, by individually replacing Glu2 and Arg10 on both peptide chains with a Leu residue, that were screened for their peroxidase-like activity. The structural and functional analysis of the best analogue, Fe(III)-Glu2Leu(TD)-MC6, indicates that an Arg residue in proximity to the heme-distal site, could assist the catalysis by favouring the formation of the Compound I, similarly to the conserved Arg38 in HRP. The main drawback of Mimochrome VI and its analogues relies in the lack of three-dimensional structures. Here, the rational design, the synthesis and the characterization of six new Mimochrome compounds with improved structural and catalytic properties is reported. Starting from Glu2Leu(TD)-MC6, Aib residues were introduced at 3 and 7 positions of the decapeptide chain, obtaining a molecule, named Mimochrome VIa. In the next stage of design, the linker length between the decapeptide chain and the porphyrin was progressively decreased: Lys9 was substituted with Orn and with Dab residues, thus obtaining two new analogues, named, respectively, Lys9Orn(D)-Mimochrome VIa and Lys9Dab(D)-Mimochrome VIa. The Aib introduction allowed the identification and separation of two regioisomers, named isomer 1 and 2, for each new analogue, demonstrating the role of Aib residues as structural constraints. The peroxidase-like activity of new developed enzymes in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), by activating H2O2, was evaluated. The functional screening highlighted that: (i) introducing Aib residues increases the stability toward bleaching; (ii) decreasing the decapeptide-porphyrin linker length increases the catalytic activity. Fe(III)-Lys9Dab(D)-Mimochrome VIa isomer 1 and 2 have been selected as the best enzymes, in terms of catalytic activities and turnover number. While being more than tenfold smaller in terms of molecular weight, the new compounds display improved enzyme-like properties, that in turn approach HRP. UV-Vis, CD, EPR and Mossbauer spectroscopies were used in combination, and demonstrated that the iron(III) coordination environment is similar to that of HRP, the natural parent enzyme, confirming the correctness of the design. The NMR spectrum of the cobalt (III) complex of Lys9Dab(D)-Mimochrome VIa isomer 2 displays a unique species, in contrast with the NMR spectrum the prototype, Co(III)-Mimochrome VI, which displays a more complex spectrum indicative of the presence of multiple species in solution. This result highlights that the improvement in the catalytic properties is accompanied by an improvement in the structural properties. The complete structural determination by NMR of Co(III)-Lys9Dab(D)-Mimochrome VIa isomer 2 is in progress, in order to obtain information for useful structure-function relationship studies.