Heterolytic OO bond cleavage: Functional role of Glu113 during bis-Fe(IV) formation in MauG

The diheme enzyme MauG utilizes H₂O₂ to perform oxidative posttranslational modification on a protein substrate. A bis-Fe(IV) species of MauG was previously identified as a key intermediate in this reaction. Heterolytic cleavage of the OO bond of H₂O₂ drives the formation of the bis-Fe(IV) intermediate. In this work, we tested a hypothesis that a glutamate residue, Glu113 in the distal pocket of the pentacoordinate heme of MauG, facilitates heterolytic OO bond cleavage, thereby leading to bis-Fe(IV) formation. This hypothesis was proposed based on sequence alignment and structural comparison with other H₂O₂-utilizing hemoenzymes, especially those from the diheme enzyme superfamily that MauG belongs to. Electron paramagnetic resonance (EPR) characterization of the reaction between MauG and H₂O₂ revealed that mutation of Glu113 inhibited heterolytic OO bond cleavage, in agreement with our hypothesis. This result was further confirmed by the HPLC study in which an analog of H₂O₂, cumene hydroperoxide, was used to probe the pattern of OO bond cleavage. Together, our data suggest that Glu113 functions as an acid-base catalyst to assist heterolytic OO bond cleavage during the early stage of the catalytic reaction. This work advances our mechanistic understanding of the H₂O₂-activation process during bis-Fe(IV) formation in MauG.