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

Abstract

The non-canoncial haem oxygenase MhuD from <i>Mycobacterium tuberculosis</i> 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.<br>

Structure–function characterization of the mono- and diheme forms of MhuD, a noncanonical heme oxygenase from Mycobacterium tuberculosis

“…The crude products of the ascorbate assay were analyzed by electrospray ionization mass spectrometry (ESI-MS) and the data (Figure S13) revealed that the major product of diheme MhuD catalysis is also mycobilin with m/z 611.25, like that of monoheme. A minor product with m/z 583.25 was detected at nearly identical relative abundance as previously reported data for the monoheme MhuD reaction, and was identified as biliverdin (37). Therefore, the products of heme degradation by diheme MhuD are highly consistent with that of monoheme in that its major products are mycobilins.…”

“…While it is well-established that monoheme MhuD degrades heme to mycobilin products (3,17,37), the enzymatic activity of diheme MhuD reported here for the first time calls for characterization of its heme degradation products. The crude products of the ascorbate assay were analyzed by electrospray ionization mass spectrometry (ESI-MS) and the data (Figure S13) revealed that the major product of diheme MhuD catalysis is also mycobilin with m/z 611.25, like that of monoheme.…”

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

“…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. 5,13 Perhaps not surprisingly, the unusual geometric and electronic structures of the heme substrate in non-canonical heme oxygenases have often been invoked to explain the novel mechanisms of these enzymes. Prior to the characterizations of the geometric and electronic structures of S. aureus IsdG and IsdI described above, it was discovered that these enzymes degrade heme to novel staphylobilin products that are distinct from the biliverdin products of canonical heme oxygenases.…”

“…19 Recently, it has been reported that both the R26S and W66F second-sphere substitutions can change the primary MhuD product from mycobilin to biliverdin. 13,20 These observations have been attributed to a mechanism where the structural and electronic dynamics of hemebound MhuD are critical: MhuD converts "ruffled" heme to meso-hydroxyheme and "planar" meso-hydroxyheme to mycobilin. The proposed monooxygenation mechanism is consistent with a recent QM/MM study of MhuD-catalyzed heme degradation.…”

“…A recently established UV/Vis absorption (Abs) spectroscopy-based assay was employed to quantify heme ruffling instead of the more laborious crystallographybased approach previously utilized for IsdI. 1,3,5 Ruffling-induced heme electronic structure changes have been invoked as a key component in non-canonical heme oxygenase mechanisms, 10,13,15,17,19,21 so a careful assessment of the heme electronic structure of these variants using 1 H NMR and MCD spectroscopies was carried out. Finally, the rates of heme monooxygenation by WT and W67F IsdG were quantified using a UV/Vis Abs-based assay and the products of these enzyme variants were identified using electrospray ionization mass spectrometry (ESI-MS).…”