Chaperonin-Nanocaged Hemin as an Artificial Metalloenzyme for Oxidation Catalysis

Taking inspiration from biology's effectiveness in functionalizing protein-based nanocages for chemical processes, we describe here a rational design of an artificial metalloenzyme for oxidations with the bacterial chaperonin GroEL, a nanocage for protein folding in nature, by supramolecular anchoring of catalytically active hemin in its hydrophobic central cavity. The promiscuity of the chaperonin cavity is an essential element of this design, which can mimic the hydrophobic binding pocket in natural metalloenzymes to accept cofactor and substrate without requiring specific ligand-protein interactions. The success of this approach is manifested in the efficient loading of multiple monomeric hemin cofactors to the GroEL cavity by detergent dialysis and good catalytic oxidation properties of the resulting biohybrid in tandem with those of the clean oxidant of H₂O₂. Investigation of the mechanism of hemin-GroEL-catalyzed oxidation of two-model substrates reveals that the kinetic behavior of the complex follows a ping-pong mechanism in both cases. Through comparison with horseradish peroxidase, the oxidative activity and stability of hemin-GroEL were observed to be similar to those found in natural peroxidases. Adenosine 5'-triphosphate (ATP)-regulated partial dissociation of the biohybrid, as assessed by the reduction of its catalytic activity with the addition of the nucleotide, raises the prospect that ATP may be used to recycle the chaperonin scaffold. Moreover, hemin-GroEL can be applied to the chromogenic detection of H₂O₂, which (or peroxide in general) is commonly contained in industrial wastes. Considering the rich chemistry of free metalloporphyrins and the ease of production of GroEL and its supramolecular complex with hemin, this work should seed the creation of many new artificial metalloenzymes with diverse reactivities.