Firpo, Vincenzo (2018) Functional Characterization of Synthetic Metalloporphyrin-Containing Enzymes. [Tesi di dottorato]

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Over the last decades, the analysis of the catalytic properties of metalloenzymes has received increasing attention, because of the variety of complex chemical transformations that these molecules are able to perform. The excellent catalytic potential of metalloenzymes derives from their aptitude to easily form highly reactive intermediates, resulting from the stabilization of metal ion oxidation states. In the effort to resemble (or even enhance) the catalytic properties of natural metalloenzymes while improving their chemical stability, a wide variety of bioinspired systems have been devised, obtained modifying either the metal cofactor or the protein matrix. In this context, the development of a novel artificial heme-enzyme belonging to the Mimochrome family (Mimochrome VIa or MC6a) and the evaluation of its catalytic potential have been explored during this PhD project. In early studies, the design, synthesis and characterization of the FeIII-containing MC6a (FeIII-MC6a) have been performed. Compared to the best candidate among previous mimochrome members, FeIII-MC6a acted as an even more efficient peroxidase-like catalyst, since it was able to oxidize ABTS (taken as model substrate) with a much higher turnover number (from 5900 to 14000) and turnover frequency (from 2300 to 5900 s-1). This feature has been correlated with the presence of two Aib residues in place of Gln3 e Ser7 on the (D) chain. Indeed, this substitution has been found to significantly increase the conformational rigidity of the peptide chain, contributing to a higher preorganization of the molecule toward protein folding. Furthermore, the catalyst was endowed with a higher robustness than that of all previous mimochrome members (up to a two-fold increase), although at the cost of a minor affinity toward H2O2 (up to a three-fold increase of KM value). In this case, it has been conjectured that the larger hydrophobic character of the (D) chain due to the presence of Aib residues may have resulted into additional interactions between the peptide chain and the porphyrin ring, thereby providing a more compact structure, which protects the metal cofactor from H2O2-mediated bleaching. In subsequent studies, the analysis of the reactivity of a FeIII-MC6a derivative containing a CoIII ion (Co-MC6a) has been conducted. These studies, which were mainly carried out under the supervision of Prof. K. L. Bren (University of Rochester, NY), were aimed to investigate the potential use of MC6a in hydrogen evolution reactions (HERs), in view of a potential employ of our catalyst in water splitting approaches. In this case, Co-MC6a is intended to represent an alternative to natural hydrogenases, whose use as catalysts for HERs is limited by their sensitivity to molecular oxygen. Co-MC6a demonstrated to be a convenient electrocatalyst in HERs. Differently from most natural enzymes or synthetic organometallic biomimetics, it acted as a water-soluble catalyst, working in neutral water and in the presence of molecular oxygen. In addition, compared to previous Co-porphyrin based synthetic enzymes (e.g. Co-MP11), Co-MC6a worked with similar turnover frequency and overpotential values but with much higher turnover numbers (up to 300000), retaining its activity even after several hours. Interestingly, the comparative analysis of Co-MP11 with Co-MC6a enabled to correlate the overpotential value in HERs with the enzyme folding. In addition, preliminary SAR studies have been undertaken, in order to identify the existing correlation between coordination shell of our enzyme and its efficacy in HERs. The result of this study will allow exploring the chemical space enabling further improvement in the enzyme-like properties of our catalyst. Overall, the results obtained during this PhD thesis represent a significant improvement in our knowledge of peptide-based artificial metalloenzymes, as they have contributed to provide the most advanced candidate with catalytic potential. Given the ease in having access to these molecules and their mutants, future endeavours will be addressed to an in-depth study of the structural requirements for enzymatic activity, in order to develop even higher performing catalysts than natural metalloenzymes. Furthermore, given the possibility to easily change the metal cofactors as well as their coordination spheres, efforts will be focused in widening the repertoire of reactions and thereby the potential applications of these enzymes.

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