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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 OO bond in MhuD-heme-OOH to the β- or δ-meso carbon of the porphyrin ligand yielding β- or δ-meso-hydroxyheme, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Non-canonical heme oxygenases are enzymes that degrade heme to non-biliverdin products within bacterial heme iron acquisition pathways. These enzymes all contain a conserved second-sphere Trp residue that is essential for enzymatic turnover. Here, UV/Vis absorption (Abs) and circular dichroism (CD) spectroscopies were employed to show that the W67F variant of IsdG perturbs the heme substrate conformation. In general, a dynamic equilibrium between "planar" and "ruffled" substrate conformations exists within non-canonical heme oxygenases, and that the second-sphere Trp favors population of the "ruffled" substrate conformation. 1 H nuclear magnetic resonance and magnetic CD spectroscopies were used to characterize the electronic structures of IsdG and IsdI variants with different substrate conformational distributions. These data revealed that the "ruffled" substrate conformation promotes partial porphyrin-to‑iron electron transfer, which makes the meso carbons of the porphyrin ring susceptible to radical attack. Finally, UV/Vis Abs spectroscopy was utilized to quantify the enzymatic rates, and electrospray ionization mass spectrometry was used to identify the product distributions, for variants of IsdG with altered substrate conformational distributions. In general, the rate of heme oxygenation by non-canonical heme oxygenases depends upon the population of the "ruffled" substrate conformation. Also, the production of staphylobilin or mycobilin by these enzymes is correlated with the population of the "ruffled" substrate conformation, since variants that favor population of the "planar" substrate conformation yield significant amounts of biliverdin. These data can be understood within the framework of a concerted rearrangement mechanism for the monooxygenation of heme to meso-hydroxyheme by non-canonical heme oxygenases., (Copyright © 2022 Elsevier Inc. All rights reserved.)