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

Authors

• Piotr Mak, Saint Louis University
• Sandra Acebes, Barcelona Supercomputing Center
• Victor Guallar, Barcelona Supercomputing Center
• James R. Kincaid, Marquette UniversityFollow
• Anabella Ivancich, Bioénergétique et Ingénierie des Protéines

Document Type

Article

Publication Date

4-2026

Publisher

Elsevier

Source Publication

Journal of Inorganic Biochemistry

Source ISSN

0162-0134

Abstract

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 Para­ magnetic 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 (v7a) 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 ad­ vantageous 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.

Comments

Published version. Journal of Inorganic Biochemistry, Vol. 277 (2026): 113199. DOI. © 2026 Elsevier. 

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Recommended Citation

Mak, Piotr; Acebes, Sandra; Guallar, Victor; Kincaid, James R.; and Ivancich, Anabella, "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" (2026). Chemistry Faculty Research and Publications. 1112.
https://epublications.marquette.edu/chem_fac/1112