De Novo Design, Synthesis and Characterisation of MP3, A New Catalytic Four-Helix Bundle Hemeprotein

Authors

  • Marina Faiella,

    Corresponding author
    1. Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Via Cintia, 80126 Naples (Italy), Fax: (+39) 081-674090
    2. Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft (The Netherlands), Fax: (+31) 15-278-2355
    • Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Via Cintia, 80126 Naples (Italy), Fax: (+39) 081-674090
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  • Dr. Ornella Maglio,

    1. Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Via Cintia, 80126 Naples (Italy), Fax: (+39) 081-674090
    2. Permanent address: IBB, CNR, Via Mezzocannone 16, 80134 Napoli (Italy)
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  • Prof. Dr. Flavia Nastri,

    1. Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Via Cintia, 80126 Naples (Italy), Fax: (+39) 081-674090
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  • Prof. Dr. Angela Lombardi,

    1. Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Via Cintia, 80126 Naples (Italy), Fax: (+39) 081-674090
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  • Dr. Liliana Lista,

    1. Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Via Cintia, 80126 Naples (Italy), Fax: (+39) 081-674090
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  • Prof. Dr. Wilfred R. Hagen,

    1. Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft (The Netherlands), Fax: (+31) 15-278-2355
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  • Prof. Dr. Vincenzo Pavone

    Corresponding author
    1. Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Via Cintia, 80126 Naples (Italy), Fax: (+39) 081-674090
    • Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Via Cintia, 80126 Naples (Italy), Fax: (+39) 081-674090
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Abstract

A new artificial metalloenzyme, MP3 (MiniPeroxidase 3), designed by combining the excellent structural properties of four-helix bundle protein scaffolds with the activity of natural peroxidases, was synthesised and characterised. This new hemeprotein model was developed by covalently linking the deuteroporphyrin to two peptide chains of different compositions to obtain an asymmetric helix–loop–helix/heme/helix–loop–helix sandwich arrangement, characterised by 1) a His residue on one chain that acts as an axial ligand to the iron ion; 2) a vacant distal site that is able to accommodate exogenous ligands or substrates; and 3) an Arg residue in the distal site that should assist in hydrogen peroxide activation to give an HRP-like catalytic process. MP3 was synthesised and characterised as its iron complex. CD measurements revealed the high helix-forming propensity of the peptide, confirming the appropriateness of the model procedure; UV/Vis, MCD and EPR experiments gave insights into the coordination geometry and the spin state of the metal. Kinetic experiments showed that FeIII–MP3 possesses peroxidase-like activity comparable to R38A–hHRP, highlighting the possibility of mimicking the functional features of natural enzymes. The synergistic application of de novo design methods, synthetic procedures, and spectroscopic characterisation, described herein, demonstrates a method by which to implement and optimise catalytic activity for an enzyme mimetic.

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