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–N 3) and cyanide-inhibited MhuD (MhuD–heme–CN). The ground state electronic structures of MhuD–heme–N 3 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 O O 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. [ABSTRACT FROM AUTHOR]