Nuclear magnetic resonance-based determination of dioxygen binding sites in protein cavities

The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O₂ gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O₂ in solvent to 8.9 mM, O₂ -induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C-terminal domain. To determine the number of O₂ binding sites and their atomic coordinates from the 1/r⁶ distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O₂ -accessible sites were identified in internal cavities: One site was consistent with the xenon-binding site in the protein in crystal, and the other site was established to be a novel ligand-binding site. MD simulations performed at 10 and 100 mM O₂ revealed dioxygen ingress and egress as well as rotational and translational motions of O₂ in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O₂ association with the internal protein cavities.