Loss of fluorine during crosslinking by the biarylitide P450_Blt proceeds due to restricted peptide orientation

The biarylitide crosslinking enzyme P450_Blt can perform crosslinking between m-F-Tyr-3 and His-5 residues within peptide substrates with concomitant and specific loss of fluorine. Our investigations suggest that a small intrinsic preference for coupling ipso to fluorine is magnified by the binding of the peptide in a specific orientation that enforces the loss of fluorine during peptide crosslinking, likely via a two-step reaction mechanism involving the non-enzyme catalysed reductive elimination of fluoride.