Lactoperoxidase: Identification of the two Tyr radical sites for the stabilization of the pH-dependent [Fe(IV) = O Tyr•] catalytic intermediates by resonance Raman and multifrequency Electron Paramagnetic Resonance spectroscopies

We have fully characterized the high-valent intermediates formed during the catalytic cycle of lactoperoxidase (LPO), a heme-containing enzyme present in milk, saliva, tears and airways, playing an important role in mammalian antimicrobial defense. Our combined approach of multifrequency (9-285 GHz) Electron Paramagnetic Resonance (EPR) and resonance Raman (rR) spectroscopies allowed the identification of Tyr21 and Tyr193 as radical sites, having a concerted role with the heme iron and resulting in two distinct [Fe(IV) = O Tyr^•] intermediates in LPO's catalytic cycle. The presence and strength of H-bonds to Tyr^• assessed by the C - O stretching mode (v_7a) and the gx component of the g-tensor detected in their rR and 285-GHz EPR spectra, respectively, induced a difference in pH-dependent stabilization of the [Fe(IV) = O Tyr₂₁^•] and [Fe(IV) = O Tyr₁₉₃^•] intermediates. The later resulted from single-step electron tunneling subsequent to the formation of the [Fe(IV) = O Por^•+] species. A Trp radical contributing to the LPO 285-GHz EPR spectra, discern by the advantageous resolution of g-values, is proposed to facilitate the electron transfer to Tyr193. Our computational analysis (PELE) crucially identified a unique surface binding site for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), close to Tyr21 (shown to host the spin by QM/MM simulations), thus consistent with LPO Tyr^• pH 5.6 detected in the rR and 285-GHz EPR spectra and the substrate oxidation previously shown by our UV-Vis stopped flow electronic absorption studies. The elucidation of the catalytic electron transfer pathways and the protein-based oxidizing sites is relevant to harnessing the concerted reactivity of heme and specific Tyr/Trp sites in natural biocatalysts.