Extended Data Fig. 6: A map of key interactions and distances in a selected view (chain F) of the active site in the Fe2(II/II)•l-Ile•PolF complex.

Chain F was selected because the electron density map for the l-Ile substrate is the most detailed and provides a basis to model the entire side chain with high confidence. Metal-ligand and hydrogen-bonding interactions are shown as dashed lines. Distances between Fe2 and potential C-H bonds targeted for H-atom abstraction are shown as solid lines. All distances are given in Å. A water molecule coordinated to Fe2 observed in other chains is shown as a dashed circle with representative bonding distance indicated. Unlike other Fe₂(II/II)-HDO structures, PolF does not exhibit two adjacent open coordination sites on each metal ion that might delineate the position of a μ-1,2-peroxo complex, indicating that conformational changes might need to occur in the first coordination sphere to accommodate O₂ addition. The coordination mode of l-Ile and observed positioning of its side chain locates C3 and C4/4’ within reasonable proximity of the diiron cluster for target by reactive intermediates. However, in this model, only C4/4’ are oriented appropriately to provide access to the relevant C-H bonds. Target of C3 would require rotation of the side chain or other conformational change. Experiments with deuterium isotopologs of l-Val indicate that all three carbon atoms can undergo HAT, but labeling at C4/4’ yields a more pronounced KIE on decay of the μ-1,2-peroxo complex, perhaps consistent with the more favorable positioning of the analogous atoms in the l-Ile complex.