Faiella, Marina (2010) Metalloprotein models: developing catalytic systems. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||metalloprotein models; de novo design; di-iron-oxo proteins; four-helix bundle; miniaturized heme proteins; peroxidase activity; bioinorganic chemistry|
|Date Deposited:||02 Dec 2010 08:50|
|Last Modified:||30 Apr 2014 19:45|
Metal–containing proteins are of particular interest: they are responsible for catalysing important biological process, such as photosynthesis, respiration, water oxidation, molecular oxygen reduction and nitrogen fixation. Understanding the structure-function relationship in protein science is an outstanding issue to disclose how macromolecules work, with the ultimate goal to reproduce or modify their activity and to design new enzymes for tailored applications.To this end, metalloprotein mimetics have been developed through the introduction of novel metal-binding sites into naturally occurring proteins as well as through de novo protein design. In this thesis, the challenge of reproducing metalloprotein active sites was approached by using a miniaturization process. The attention was centered on natural iron-containing proteins and on artificial models called DFs and Mimochromes, which encompass the structural and functional features of di-iron-oxo proteins and heme-proteins, respectively. Both classes of miniaturized models demonstrate the importance of considering numerous structural issues in protein design, to obtain soluble and stable molecules, and to optimize metal-binding sites for function. This thesis reports the functional and chemico-physical properties of DF3, a de novo designed di-iron protein model. DF3 is the latest member of the DF family of synthetic proteins. They consist of helix–loop–helix hairpins, designed to dimerize and form an antiparallel four-helix bundle that encompasses a metal-binding site similar to those of non-heme carboxylate-bridged di-iron proteins. All the spectroscopic and structural features reported herein demonstrate the ability of a designed protein to finely tune the active-site structure to accommodate different metal ions and exogenous ligands. Further, the complete analysis of the di-iron complex supports the ability of di-Fe(III)–DF3 to catalyze oxidation reactions, in particular of phenols to quinines. To confirm the structural basis for DF3 catalytic activity, its NMR solution structure was solved using the diamagnetic di-Zn(II) derivative. Then, structural features of DFs and natural di-iron proteins, as well as functional elements of Mimochromes and natural horseradish peroxidase, were borrowed to obtain a de novo protein class with five-coordinated heme-complex and peroxidase activity, named MPs (Mini-Peroxidases). The basic structure of these models consists in a deuterohemin covalently linked to two helix-loop-helix peptide chains. The active site presents (i) an homo-Cys/His residue in a single chain that acts as axial ligand to the iron ion, leaving the sixth coordination site able to accommodate exogenous ligands or substrates; (ii) an Arg residue in the distal site that should be able to activate hydrogen peroxide to give HRP-like catalytic process. The last analogue MP3 was partially characterized as the iron ion species, and its biocatalytic efficiency respect to natural systems was evaluated. In summary, this thesis reports the last achievements in protein design of di-iron-oxo-proteins with desired catalytic activity; it also reports design, synthesis and characterization of a new class of peptide-based heme-models, named MPs, as well as preliminary catalytic activity studies.
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