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

Abstract

The noncanonical 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 refined enzymatic mechanism. UV/vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring the population of the ruffled heme conformation changed the rate-limiting step of the reaction, resulting in a measurable buildup 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 α-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 heme 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 refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.

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

“…It was previously shown that the major products of WT IsdG and WT MhuD are staphylobilin and mycobilin, respectively [13,17]. Recently, it was realized that a second-sphere variant of MhuD that decreases population of the "ruffled" conformation, W66F, changes the major product of MhuD from mycobilin to biliverdin [20]. The ESI-MS data reported here reveals that decreased population of the "ruffled" conformation of IsdG induced by the W67F substitution also increases production of biliverdin (Fig.…”

“…It turns out that MhuD degrades heme to a third product, mycobilin [17], via sequential monooxygenation and dioxygenation steps [18]. Recently, it has been reported that both the R26S and W66F second-sphere substitutions can change the primary MhuD product from mycobilin to biliverdin [19,20]. These observations have been attributed to a mechanism where the structural and electronic dynamics of heme-bound MhuD are critical: MhuD converts "ruffled" heme to meso-hydroxyheme and "planar" meso-hydroxyheme to mycobilin.…”

“…A recently established UV/Vis absorption (Abs) spectroscopy-based assay was employed to quantify heme ruffling instead of the more laborious crystallography-based approach previously utilized for IsdI [1,3,5]. Rufflinginduced heme electronic structure changes have been invoked as a key component in non-canonical heme oxygenase mechanisms [10,14,16,18,20,21], 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 oxygenation by WT and W67F IsdG were quantified using a UV/Vis Absbased assay and the products of these enzyme variants were identified using electrospray ionization mass spectrometry (ESI-MS) [15].…”

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

“…It turns out that MhuD degrades heme to a third product, mycobilin [17], via sequential monooxygenation and dioxygenation steps [18]. Recently, it has been reported that both the R26S and W66F second-sphere substitutions can change the primary MhuD product from mycobilin to biliverdin [19,20]. These observations have been attributed to a mechanism where the structural and electronic dynamics of heme-bound MhuD are critical: MhuD converts "ruffled" heme to meso-hydroxyheme and "planar" meso-hydroxyheme to mycobilin.…”

“…A recently established UV/Vis absorption (Abs) spectroscopy-based assay was employed to quantify heme ruffling instead of the more laborious crystallography-based approach previously utilized for IsdI [1,3,5]. Rufflinginduced heme electronic structure changes have been invoked as a key component in non-canonical heme oxygenase mechanisms [10,14,16,18,20,21], 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 oxygenation by WT and W67F IsdG were quantified using a UV/Vis Absbased assay and the products of these enzyme variants were identified using electrospray ionization mass spectrometry (ESI-MS) [15].…”