The enzyme known as dehaloperoxidase-hemoglobin (DHP) has provided us with a recent example of a multifunctional protein that challenges many of the assumptions behind the structure-function correlation. Of course, structure is related to function, but the question is whether, and how, structure uniquely defines function. DHP appears to suggest that the substrate itself plays a pivotal role in determining which enzyme activity it performs. Termed the Substrate Directed EnzymatiC Activity paRadigm (“SiDECAR”), this plasticity of chemical reactivity in DHP can be used to interrogate protein-structure function relationships, enabling us to explore in detail how Nature controls the selectivity of function across the heme protein superfamily. Originally identified as a peroxidase in an oxidative dehalogenation reaction that utilizes H2O2 to convert trihalophenols to dihaloquinones, we have further explored the substrate scope of this catalytic globin using haloindoles, nitrophenols, and pyrroles. Using a combination of spectroscopic techniques paired with biochemical assays and crystallography (X-ray and neutron), we have now shown that DHP possesses both peroxygenase and oxidase activities using haloindoles and nitrophenols as new classes of substrates. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope 18O-labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H218O2, indicative of a previously unreported peroxygenase activity for DHP. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded 5,5’-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, with O2-consumption studies and non-enzymatic controls together suggesting a newly identified oxidase activity for DHP. Preliminary data also suggest that DHP is able to catalyze the oxidation of 2,3-dimethylindole by a previously unreported oxygenase mechanism that is reminiscent of lipoxygenase, a non-heme dioxygenase. Thus, DHP provides an unprecedented platform for probing mechanistic questions related to the protein environment: by simply changing the substrate, we can investigate the unique set of protein-substrate interactions related to each of the five different heme activities (O2-transport, peroxidase, peroxygenase, oxidase and oxygenase) of DHP, enabling us to pose questions related to multiple activities using just this single enzyme.

Mechanistic, structural and computational studies of the catalytic multifunctional hemoglobin dehaloperoxidase from Amphitrite ornata

253rd National Meeting (2017)

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