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 O 2 gas‐pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O 2 in solvent to 8.9 mM, O 2 ‐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 O 2 binding sites and their atomic coordinates from the 1/ r 6 distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid‐search. Two O 2 ‐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 O 2 revealed dioxygen ingress and egress as well as rotational and translational motions of O 2 in the cavities. It is therefore suggested that conformational fluctuations within the ground‐state ensemble transiently develop channels for O 2 association with the internal protein cavities.