An artificial heme-enzyme with enhanced catalytic activity: evolution, functional screening and structural...

Area of Science:

• Biochemistry
• Synthetic Biology
• Protein Engineering

Background:

• Synthetic proteins offer minimal, well-defined scaffolds to replicate metalloprotein functions.
• The Mimochrome family of heme-protein models was initially designed to mimic bis-His cytochrome b.

Purpose of the Study:

• To rationally refine the function of Mimochrome heme-protein models, introducing and enhancing peroxidase-like activity.
• To explore modifications for improved catalytic activity in synthetic heme-protein scaffolds.

Main Methods:

• Modification of the Mimochrome structure to create a distal cavity near the heme.
• Introduction of single amino acid substitutions in Mimochrome VI (MC6) to generate new variants.
• Screening of modified compounds for peroxidase activity and detailed structural/functional analysis of the best analogue.

Main Results:

• Successful introduction of peroxidase-like activity in a modified Mimochrome system (Mimochrome VI).
• Identification of Fe(III)-E(2)L(TD)-MC6 as the best-performing analogue.
• Demonstration that an arginine residue near the heme-distal site enhances catalysis by promoting the formation of the "compound I" intermediate, mimicking horseradish peroxidase (HRP).

Conclusions:

• Small, well-defined scaffolds can be effectively used to investigate factors influencing heme-protein activity.
• The study highlights the potential for programming new functions into synthetic proteins.
• The findings provide insights into the role of specific amino acid residues in tuning enzyme catalysis.