DESIGNING A MOLYBDENUM CONTAINING ARTIFICIAL METALLOENZYME WITH A SYNTHETIC PTERIN COFACTOR

Abstract

Molybdenum is an essential second row transition metal found at the active site of the enzymes that play critical role in redox reactions involved in the global nitrogen, carbon and sulfur cycle. These enzymes utilize a unique pterin based cofactor called pyranopterin to coordinate molybdenum and facilitate oxygen atom transfer chemistry. Despite continuous research, many of the mechanistic details remain elusive because native molybdenum enzymes present substantial experimental challenges including biosynthesis of cofactor, oxygen sensitivity of the reduced molybdenum center and structurally rigid active sites inaccessible to chemical modification. Artificial metalloenzymes (ArMs) provide a unique approach to overcome these limitations by assembling a synthetic metal-cofactor complex within a protein scaffold. This approach enables systematic investigation of metal ligand interaction and catalytic function in a chemically accessible environment. Herein, I describe my efforts toward the design and assembly of streptavidin-based molybdenum containing artificial metalloenzyme. In this research, leucopterin was selected as a synthetic cofactor due to its structural similarity to the natural pyranopterin cofactor. Leucopterin was covalently conjugated to biotin through an amide bond using a pentafluorophenyl ester to facilitate the conjugation, forming biotinylated leucopterin as a bifunctional molecule capable of both binding tightly to streptavidin, anchoring the cofactor, and molybdenum coordination. Structural characterization by ¹H and ¹³C NMR spectroscopy confirmed successful amide bond formation and intact retention of both the leucopterin pteridine framework and the biotin scaffold. HABA displacement assay confirmed competitive displacement of HABA from the streptavidin binding pocket by the biotinylated leucopterin molybdate complex. The assembled streptavidin based biotinylated leucopterin molybdate complex was characterized by EPR spectroscopy. There was a detectable Mo (V) signal upon addition of dithionite, providing the direct spectroscopic evidence of molybdenum reduction and Mo pterin redox interaction in this system. Together the results from this research work represent a foundational work for future detailed structural characterization, optimization of Mo incorporation conditions and evaluation of catalytic oxygen atom transfer activity in this artificial molybdoenzyme system.