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

Abstract

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

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

“…30 Kinetic analysis is complicated by the fact that IsdG intermediates and products absorb light at this wavelength, 16 but these issues have been addressed in a recently reported analytical expression. 13 Based upon the data reported here (Figure 7), and the kinetic model reported previously, WT IsdG monooxygenates heme with a pseudo-first order rate constant of 0.111 ± 0.004 min -1 . Upon introduction of the W67F substitution, the rate decreases to 0.049 ± 0.006 min -1 .…”

“…14 However, a recent in proteo MS study of the non-canonical HO MhuD revealed that the product isolation approach can fail to detect alternate enzyme products. 13 Indeed, ESI-MS of the WT IsdG reaction mixture 60 min after the addition of ascorbate reveals a mixture of m/z 583, 599, and 611 species (Figure 8). The 599 and 611 Da species were previously observed for IsdG-catalyzed heme degradation, 16 and attributed to the staphylobilin product and formyloxobilin intermediate, respectively.…”

“…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).…”