O2 reactivity of flavoproteins: Dyamic access of dioxygen to the active site and role of a H+ relay system in D-amino acid oxidase

Molecular dynamics simulations and implicit ligand sampling methods have identified trajectories and sites of high affinity for O2 in the protein framework of the flavoprotein D-amino-acid oxidase (DAAO). A specific dynamic channel for the diffusion of O2 leads from solvent to the flavin Si-side (amino acid substrate and product bind on the Re-side). Based on this, amino acids that flank the putative O2 high affinity sites have been exchanged with bulky residues to introduce steric constraints. In G52V DAAO, the valine side chain occupies the site that in wild-type DAAO has the highest O2 affinity. In this variant, the reactivity of the reduced enzyme with O2 is decreased ≥100-fold and the turnover number ≈1000-fold thus verifying the concept. In addition, the simulations have identified a chain of three water molecules that might serve in relaying a H+ from the product imino acid =NH++ group bound on the flavin Reside to the developing peroxide on the Si-side. This function would be comparable with that of a similarly located histidine in the flavoprotein glucose oxidase.