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

Abstract

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 O-O 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 O-O 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 characterization of the reaction between MauG and H2O2 revealed that mutation of Glu113 inhibited heterolytic O-O 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 O-O bond cleavage. Together, our data suggest that Glu113 functions as an acid-base catalyst to assist heterolytic O-O 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.

Radical Trapping Study of the Relaxation of bis-Fe(IV) MauG

“…MauG was prepared as described previously [1, 2, 4, 7, 9, 27]. Briefly, P. denitrificans cells carrying a plasmid for expression of MauG were grown in mineral salts medium at 30°C in 4 stages: 10 ml, 100 ml, 1 L, and 10 L. Tetracycline at 2 μg/ml was used for antibiotic selection.…”

“…Cell lysate was clarified by centrifugation, and the supernatant was collected. The His 6 -tagged MauG was purified by nickel affinity chromatography, desalted to remove excess imidazole, and concentrated by ultrafiltration as described previously [7, 9, 27]. All reactions were carried out in 50 mM potassium phosphate buffered to pH 9.0 for optimized intermediate production.…”

Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases

“…At the active site, two conserved residues, Glu118 and Gln108, are proposed to play an important role in the formation and stabilization of the oxoferryl (Fe 4+ -oxo) intermediate species and Glu118, in particular, in the cleavage of the hydrogen peroxide O-O bond. An identical role was proposed for a conserved glutamate residue in the heme cavity of MauG proteins, which has a similar heme cavity to BCCPs [ 35 , 36 , 37 ]. The importance of these two residues in catalysis was proven by site-directed mutagenesis studies, which showed that these single residue variants were inactive [ 20 ].…”