Nature of the Reactive Biferric Peroxy Intermediate P′ in the Arylamine Oxygenases and Related Binuclear Fe Enzymes

Binuclear nonheme iron enzymes activate O₂ to perform a wide range of chemical transformations. The process of O₂ activation typically involves a biferric peroxy-level intermediate P. It has been previously found that this intermediate undergoes further activation, either protonation or rearrangement to form P' or further oxidation to form high-valent intermediates Q or X. This study defines the structure of the P' intermediate in the <i>N</i>-oxygenases CmlI (and AurF based on previous data) using nuclear resonance vibrational spectroscopy (NRVS) in conjugation with density functional theory (DFT) calculations. These results, combined with variable temperature variable field (VTVH) magnetic circular dichroism (MCD) spectroscopy on the 1-electron cryoreduced P', define the structure of the P' intermediate as a μ-1,2-hydroxoperoxo biferric site with a second hydroxide bridge. Reaction coordinate calculations demonstrate that single electron transfer (SET) is facilitated by protonation of the peroxo, activating its reductive cleavage, and that the additional hydroxide bridge does not impact this reaction. VTVH MCD studies further reveal that the hydroxide bridge is absent in the biferrous site, suggesting that during the O₂ reaction with the biferrous site, a water molecule forms the hydroxide bridge in providing the proton that activates the peroxide in P' for reactivity.