Structural Insights into the Mechanism of a Vanadium-Dependent Bromoperoxidase Enabled by High-Resolution Cryo-EM

Vanadium-dependent haloperoxidases (VHPOs) are a uniquely robust class of enzymes capable of performing electrophilic halogenation of organic substrates. Despite their emerging biotechnological significance, the precise catalytic and halide-selection mechanisms of VHPOs under native conditions remains largely unexamined. Herein, we leverage single-particle cryogenic transmission electron microscopy (cryo-EM) to provide insight into the catalytic mechanism of the vanadium-dependent bromoperoxidase from Corallina pilulifera (CpVBPO) under catalytically relevant conditions. Specifically, we have collected the first set of high-resolution cryo-EM structures of CpVBPO in four catalytically relevant states including binding to phosphate, orthovanadate, orthovanadate and bromide, and orthovanadate and hydrogen peroxide. The collected structures, resolved at 2.2 to 3.2 Å, reveal deviations from previously reported X-ray diffraction (XRD) structures, particularly in the geometry of the vanadate cofactor and key residue interactions in the active site. Our findings suggest a trigonal planar, metavanadate-like geometry for the vanadate cofactor in the resting state, contradicting the commonly proposed orthovanadate geometry containing an apical hydroxo ligand. Furthermore, the dynamic roles of residues Asp335 and Leu337 in halide specificity in the catalytic cycle are proposed through observed conformational changes upon bromide binding. Using pKa calculations and structural analyses, a mechanism for generating hypobromous acid, mediated by His487, is presented. This study not only refines the structural and mechanistic understanding of CpVBPO, but also demonstrates the capabilities of cryo-EM to capture native-like enzyme structures under catalytically relevant conditions, offering a more informative approach for investigating the mechanistic features of VHPOs.