Second-sphere tuning of analogues for the ferric-hydroperoxoheme form of Mycobacterium tuberculosis MhuD

Mycobacterium tuberculosis MhuD catalyzes the oxygenation of heme to mycobilin; experimental data presented here elucidates the novel hydroxylation reaction catalyzed by this enzyme. Analogues for the critical ferric–hydroperoxoheme (MhuD–heme–OOH) intermediate of this enzyme were characterized using UV/Vis absorption (Abs), circular dichroism (CD), and magnetic CD (MCD) spectroscopies. In order to extract electronic transition energies from these spectroscopic data, a novel global fitting model was developed for analysis of UV/Vis Abs, CD, and MCD data. A variant of MhuD was prepared, N7S, which weakens the affinity of heme-bound enzyme for a hydroperoxo analogue, azide, without significantly altering the protein secondary structure. Global fitting of spectroscopic data acquired in this study revealed that the second-sphere N7S substitution perturbs the electronic structure of two analogues for MhuD–heme–OOH: azide-inhibited MhuD (MhuD–heme–N3) and cyanide-inhibited MhuD (MhuD–heme–CN). The ground state electronic structures of MhuD–heme–N3 and MhuD–heme–CN were assessed using variable-temperature, variable-field MCD. Altogether, these data strongly suggest that there is a hydrogen bond between the Asn7 side-chain and the terminal oxygen of the hydroperoxo ligand in MhuD–heme–OOH. As discussed herein, this finding supports a novel hydroxylation reaction mechanism where the Asn7 side-chain guides a transient hydroxyl radical derived from homolysis of the OO bond in MhuD–heme–OOH to the β- or δ-meso carbon of the porphyrin ligand yielding β- or δ-meso-hydroxyheme, respectively. Synopsis: In this work, it is shown that Mycobacterium tuberculosis MhuD utilizes a hydrogen bond between Asn7 and the terminal oxygen of a hydroperoxo ligand to orient hydroperoxo along the β/δ-axis of heme. This orientation promotes regiospecific hydroxylation of heme to β- and δ-meso-hydroxyheme. [Display omitted] •Asn7 forms a hydrogen bond with the terminal atom of the distal ligand to heme in MhuD.•The hydrogen bond between Asn7 and the distal ligand orients the ligand within the active site.•A hydrogen bond with Asn7 perturbs the electronic structures of analogues for the ferric–hydroperoxoheme intermediate.•Asn7 guides a transient hydroxyl radical to the β- and δ-meso carbons of heme.•A novel global fitting method was developed for the analysis of UV/Vis Abs, CD, and MCD data.