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 Tyr ● microenvironment, i.e. H-bonding interactions 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 Tyr 21 ● and Fe(IV) = O Tyr 193 ● intermediates. The later result from single-step electron tunneling subsequent to the formation of the Fe(IV) = O Por ●+ species. A Trp radical contributing to the 285-GHz EPR spectra of LPO, 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 binding site for ABTS on LPO's surface, 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 ABTS 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.