Abstract
MhuD is a noncanonical heme oxygenase (HO) from Mycobacterium tuberculosis (Mtb) that catalyzes unique heme degradation chemistry distinct from canonical HOs, generating mycobilin products without releasing carbon monoxide. Its crucial role in the Mtb heme uptake pathway has identified MhuD as an auspicious drug target. MhuD is capable of binding either one or two hemes within a single active site, but only the monoheme form was previously reported to be enzymatically active. Here we employed resonance Raman (rR) spectroscopy to examine several factors proposed to impact the reactivity of mono- and diheme MhuD, including heme ruffling, heme pocket hydrophobicity, and amino acid–heme interactions. We determined that the distal heme in the diheme MhuD active site has negligible effects on both the planarity of the His-coordinated heme macrocycle and the strength of the Fe-NHis linkage relative to the monoheme form. Our rR studies using isotopically labeled hemes unveiled unexpected biomolecular dynamics for the process of heme binding that converts MhuD from mono- to diheme form, where the second incoming heme replaces the first as the His75-coordinated heme. Ferrous CO-ligated diheme MhuD was found to exhibit multiple Fe-C-O conformers, one of which contains catalytically predisposed H-bonding interactions with the distal Asn7 residue identical to those in the monoheme form, implying that it is also enzymatically active. This was substantiated by activity assays and MS product analysis that confirmed the diheme form also degrades heme to mycobilins, redefining MhuD’s functional paradigm and further expanding our understanding of its role in Mtb physiology.
Highlights
Degradation of heme is a critically important physiological process in many forms of life catalyzed by heme oxygenases (HOs)
These results indicate that MhuD contains a predominantly hydrophobic distal active site environment
Structure-function correlation of mono- and diheme MhuD. resonance Raman (rR) spectroscopy was employed in this study to investigate several features of the active site environment that dictate the reactivity of mono- and diheme MhuD such as hemeplanarity, hydrophobicity of the distal active site, strength of the Feproximal ligand bond, and active site amino acid interactions with heme distal ligands and peripheral groups
Summary
Degradation of heme is a critically important physiological process in many forms of life catalyzed by heme oxygenases (HOs). Asn-7 was suggested to be key to the enzymatic activity of MhuD by providing H-bonding interactions to stabilize and regiospecifically orient reactive intermediates in the monoheme form [3], an interaction that structural data suggested to be blocked by the distal heme in diheme MhuD [10]. The initial studies showed that the monoheme form was enzymatically active, while diheme was inactive [10] This brought into question the functional role of the second heme in the catalysis of MhuD. More recent studies showed that MhuD did not evolve to preferentially bind one heme molecule and that the diheme form is comparably favored, implicating a more complex functional role of diheme MhuD in Mtb physiology [19]. RR studies of ferrous carbonmonoxy adducts of heme proteins provide an effective probe of the polarity and crowding of the distal active site environment [26, 27], making the CO adduct ideal for monitoring the MhuD heme pocket in the presence or absence of an additional heme
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