Selective inactivation of serine proteases by nonheme iron complexes

Oxidative inactivation of the serine proteases trypsin and chymotrypsin by nonheme iron complexes is described. The nonheme ligands N4Py (1) and derivative 3CG-N4Py (2), which contains a pendant guanidinium group, were used as ligands for iron. Ferryl (Fe^IVO) species derived from these ligands, [Fe ^IV(O)(N4Py)]²⁺ (7) and [Fe^IV(O)(3CG-N4Py)] ³⁺ (8), inactivate trypsin and chymotrypsin by the oxidation of amino acid side chains. Ferryl 8 is most effective with chymotrypsin (IC₅₀ value of 26 μM for 8 vs 119 μM for 7). IC₅₀ values of 71 and 54 μM were obtained for trypsin with 7 and 8, respectively. Amino acid analysis confirmed that residues cysteine, tyrosine, and tryptophan are oxidized under these conditions. Trypsin is inactivated preferentially over chymotrypsin under catalytic conditions, where the enzyme was pulsed with H ₂O₂ in the presence of ferrous complexes [Fe ^II(OH₂)(N4Py)]²⁺(5) and [Fe^II(Cl) (3CG-N4Py)]²⁺ (6). Control experiments support the action of a unique oxidant, other than ferryls or hydroxyl radicals, under these conditions, where tyrosine residues are targeted selectively.