Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation

Abstract

Bacterial pathogens must acquire host iron for survival and colonization. Because free iron is restricted in the host, numerous pathogens have evolved to overcome this limitation by using a family of monooxygenases that mediate the oxidative cleavage of heme into biliverdin, carbon monoxide, and iron. However, the etiological agent of tuberculosis, Mycobacterium tuberculosis, accomplishes this task without generating carbon monoxide, which potentially induces its latent state. Here we show that this unusual heme degradation reaction proceeds through sequential mono- and dioxygenation events within the single active center of MhuD, a mechanism unparalleled in enzyme catalysis. A key intermediate of the MhuD reaction is found to be meso-hydroxyheme, which reacts with O2 at an unusual position to completely suppress its monooxygenation but to allow ring cleavage through dioxygenation. This mechanistic change, possibly due to heavy steric deformation of hydroxyheme, rationally explains the unique heme catabolites of MhuD. Coexistence of mechanistically distinct functions is a previously unidentified strategy to expand the physiological outcome of enzymes, and may be applied to engineer unique biocatalysts.

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b

“…38 The heme oxygenase reaction is likewise autocatalytic, with ferrous heme b acting concurrently as the substrate and cofactor. In the proposed mechanism for MhuD, the IsdG-family member from Mycobacteria , 13 heme b reacts with O 2 to form a ferrous heme-O 2 complex. Addition of H + and an electron leads to a ferric-hydroperoxy intermediate, which hydroxylates one of the pyrrole-bridging meso -carbons.…”

“…This mechanism is analogous to the heme oxygenase reaction, which begins with an O 2 -mediated hydroxylation of the porphyrin ring. 12, 13 Finally, the enzyme might catalyze a reaction involving Fe(II) and H 2 O 2 (Scheme 1C). While not completely unprecedented, 14, 15 such a reaction would be highly unusual since hemes generally react with H 2 O 2 when the iron is in the ferric oxidation state.…”

Reaction intermediates in the heme degradation reaction by HutZ from Vibrio cholerae

“…11–13 Recently, an IsdG-type enzyme from Mycobacterium tuberculosis is shown to degrade heme by sequential mono- and dioxygenation reactions in contrast to the canonical three monoxygenation mechanisms of HO. 14 The mechanistic diversity prompted us to examine whether HutZ catalyzes the HO-type heme degradation in spite of their structural dissimilarity. In this study, we have characterized key reaction intermediates of HutZ, including oxyferrous heme, meso -hydroxyheme and verdoheme using UV-visible absorption and resonance Raman spectroscopy, thereby determining its heme-degradation mechanism.…”

The structure ofMycobacterium tuberculosisheme-degrading protein, MhuD, in complex with product

“…Unsurprisingly, the two distinct classes of heme-degrading enzymes degrade heme by different mechanisms (Matsui et al, 2016). Although heme is coordinated by a proximal His residue in both HO and IsdG-type enzymes, the heme molecule in HOs is near-planar, while for IsdG-type proteins, the heme is ruffled (Schuller et al, 1999;Wu et al, 2005).…”

“…In contrast, the distal heme pocket is quite hydrophobic for IsdGtype proteins, with only one or two ordered waters observed in the proximal pocket (Graves et al, 2014;Wu et al, 2005). In MhuD, it has been proposed that the hydrophobic pocket together with the ruffled heme contributes to the sequential monooxygenase and dioxygenase steps necessary for MhuD to degrade heme (Matsui et al, 2016).…”