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Bromoperoxidases from the brown alga Ascophyllum nodosum, abbreviated as V(Br)PO(AnI) and V(Br)PO(AnII), show 41% sequence homology and differ by a factor of two in the percentage of α-helical secondary structures. Protein monomers organize into homodimers for V(Br)PO(AnI) and hexamers for V(Br)PO(AnII). Bromoperoxidase II binds hydrogen peroxide and bromide by approximately one order of magnitude stronger than V(Br)PO(AnI). In oxidation catalysis, bromoperoxidases I and II turn over hydrogen peroxide and bromide similarly fast, yielding in morpholine-4-ethanesulfonic acid (MES)-buffered aqueous tert-butanol (pH 6.2) molecular bromine as reagent for electrophilic hydrocarbon bromination. Alternative compounds, such as tribromide and hypobromous acid are not sufficiently electrophilic for being directly involved in carbon-bromine bond formation. A decrease in electrophilicity from bromine via hypobromous acid to tribromide correlates in a frontier molecular orbital (FMO) analysis with larger energy gaps between the π-type HOMO of, for example, an alkene and the σ*(Br,X)-type LUMO of the bromination reagent. By using this approach, the reactivity of substrates and selectivity for carbon-bromine bond formation in reactions mediated by vanadate-dependent bromoperoxidases become predictable, as exemplified by the synthesis of bromopyrroles occurring naturally in marine sponges of the genera Agelas, Acanthella, and Axinella.

X-ray diffraction of native bromoperoxidase II (EC 1.11.1.18) from the brown alga Ascophyllum nodosum reveals at a resolution of 2.26 Å details of orthovanadate binding and homohexameric protein organization. Three dimers interwoven in contact regions and tightened by hydrogen-bond-clamped guanidinium stacks along with regularly aligned water molecules form the basic structure of the enyzme. Intra- and intermolecular disulfide bridges further stabilize the enzyme preventing altogether the protein from denaturing up to a temperature of 90 °C, as evident from dynamic light scattering and the on-gel ortho-dianisidine assay. Every monomer binds one equivalent of orthovanadate in a cavity formed from side chains of three histidines, two arginines, one lysine, serine, and tryptophan. Protein binding occurs primarily through hydrogen bridges and superimposed by Coulomb attraction according to thermochemical model on density functional level of theory (B3LYP/6-311++G**). The strongest attractor is the arginine side chain mimic N-methylguanidinium, enhancing in positive cooperative manner hydrogen bridges toward weaker acceptors, such as residues from lysine and serine. Activating hydrogen peroxide occurs in the thermochemical model by side-on binding in orthovanadium peroxoic acid, oxidizing bromide with virtually no activation energy to hydrogen bonded hypobromous acid.

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