title: De novo design, synthesis and characterisation of MP3, a new catalytic four-helix bundle hemeprotein.
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 Fe(III)-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.
authors.affiliation: Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Via Cintia, 80126 Naples, Italy. marina.faiella@unina.it
authors.forename: Marina
authors.initials: M
authors.lastname: Faiella
authors.forename: Ornella
authors.initials: O
authors.lastname: Maglio
authors.forename: Flavia
authors.initials: F
authors.lastname: Nastri
authors.forename: Angela
authors.initials: A
authors.lastname: Lombardi
authors.forename: Liliana
authors.initials: L
authors.lastname: Lista
authors.forename: Wilfred R
authors.initials: WR
authors.lastname: Hagen
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Pavone
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D005290:Ferric Compounds / Q000138:chemical synthesis* / Q000737:chemistry*; D006420:Hemeproteins / Q000138:chemical synthesis* / Q000737:chemistry*; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000138:chemical synthesis* / Q000737:chemistry*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005290:Ferric Compounds; D006420:Hemeproteins; D008667:Metalloproteins; D007501:Iron; D010544:Peroxidases