Spectroscopic Evidence for Electronic Control of Heme Hydroxylation by IsdG

Conger, Matthew; Cornetta, Amanda; Liptak, Matthew D.

doi:10.1021/acs.inorgchem.9b02530

Cited by 17 publications

(39 citation statements)

References 64 publications

(215 reference statements)

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“…10 Instead, the monooxygenation rate enhancement is likely derived from an electronic structure change similar to that previously reported for IsdG. 32 These data elucidate a mechanism where a dynamic equilibrium between planar and ruffled heme exists within the MhuD active site, 12 but heme monooxygenation only proceeds for the ruffled conformation.…”

Section: Discussionsupporting

confidence: 72%

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

Thakuri¹,

O'Rourke²,

Graves³

et al. 2020

Preprint

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The non-canoncial heme oxygenase MhuD from Mycobacterium tuberculosis binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a revised enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a revised enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.

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Section: Discussionsupporting

confidence: 72%

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

Thakuri¹,

O'Rourke²,

Graves³

et al. 2020

Preprint

Self Cite

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show abstract

“…10 Instead, the monooxygenation rate enhancement is likely derived from an electronic structure change similar to that previously reported for IsdG. 32 These data elucidate a mechanism where a dynamic equilibrium between planar and ruffled haem exists within the MhuD active site, 12 but haem monooxygenation only proceeds for the ruffled conformation.…”

Section: A Proposed Mechanism For Haem Oxygenation By Mhudsupporting

confidence: 72%

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

Thakuri¹,

O'Rourke²,

Graves³

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

The non-canoncial haem oxygenase MhuD from Mycobacterium tuberculosis binds a haem substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between haem substrate dynamics and MhuD-catalysed haem degradation resulting in a revised enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionisation mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favouring population of the ruffled haem conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyhaem intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favoured the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of haem monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyhaem dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid haem monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered haem monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyhaem. Altogether, these data yielded a revised enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into haem yielding mycobilin.

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“…5 An X-ray crystal structure for WT IsdG-heme-CN is not available, but heme ruffling in WT IsdG-heme-CN has been estimated to be 2.0 Å based upon spectroscopically-validated QM/MM calculations. 7 The UV/Vis Abs spectra of WT and W67F IsdG-heme-CN are quite similar (Figure 3), but closer inspection reveals that the W67F substitution blueshifts both the Q and Soret bands by 100 cm -1 . Based upon the QM/MM structure of WT IsdG-heme-CN, the UV/Vis Abs spectra of WT and W67F IsdG-heme-CN, and the established ruffling model noted above, the magnitude of heme ruffling in W67F IsdG-heme-CN was estimated to be 1.7 Å.…”

Section: Figurementioning

confidence: 92%

“…6 Most recently, spectroscopically-validated hybrid quantum mechanics/molecular mechanics (QM/MM) calculations suggested that S. aureus IsdG, a third non-canonical heme oxygenase, induces an intermediate degree of heme ruffling compared to IsdI and MhuD (Figure 1). 7 The observation of two distinct heme conformations within the MhuD active site is fairly unique within the heme protein literature, and the possibility that IsdG and IsdI also stabilize two distinct heme substrate conformations had not been evaluated prior to this work.…”

mentioning

confidence: 99%

“…WT IsdG induces an out-of-plane ruffling distortion of its heme substrate based upon spectroscopically-validated QM/MM calculations (top). 7 The W67F substitution was hypothesized to decrease heme ruffling by reducing steric interactions between residue 67 and the substrate (middle). This heme structural change would be expected to change the ground state electronic configuration from 2 B2g to 2 Eg by decreasing mixing of the Fe 3dxy and porphyrin a2u orbitals (bottom) resulting in altered heme oxygenation activity.…”

mentioning

confidence: 99%

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Ruffling is Essential for Staphylococcus aureus IsdG-catalyzed Degradation of Heme to Staphylobilin

Schuelke-Sanchez

Cornetta

Kocian

et al. 2021

Preprint

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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. Previous studies have revealed several important roles for the conserved second-sphere Trp in Staphylococcus aureus IsdG, S. aureus IsdI, and Mycobacterium tuberculosis MhuD. However, a general model for the geometric, electronic, and functional role of the second-sphere Trp had not been deduced prior to this work. 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 without altering the protein secondary structure. In general, it can now be stated that a dynamic equilibrium between "planar" and "ruffled" substrate conformations exists within non-canonical heme oxygenases, and that the secondsphere 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 monooxygenation to meso-hydroxyheme by non-canonical heme oxygenasess 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. However, the mechanisms of IsdG/IsdI and MhuD must diverge following this intermediate in order to generate distinct staphylobilin and mycobilin products, respectively.

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Spectroscopic Evidence for Electronic Control of Heme Hydroxylation by IsdG

Cited by 17 publications

References 64 publications

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

Ruffling is Essential for Staphylococcus aureus IsdG-catalyzed Degradation of Heme to Staphylobilin

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