Crystallographic and Spectroscopic Insights into Heme Degradation by Mycobacterium tuberculosis MhuD

Abstract

Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD–heme–CN) as well as detailed 1H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature 1H NMR spectrum of MhuD–heme–CN is consistent with population of both a 2Eg electronic state with a (dxy)2(dxz,dyz)3 electron configuration, similar to the ground state of canonical HOs, and a 2B2g state with a (dxz,dyz)4(dxy)1 electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD–heme–CN has a 2B2g electronic ground state with a low-lying 2Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD–heme–CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon.

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

“…Inefficiency of monooxygenation is evident both in the H 2 O 2 - (Fig. S4) and O 2 /e -reactions of MhuD (14), probably due to its predominantly hydrophobic heme environment lacking a protondonating candidate to control the reactive FeOOH intermediate (16,30). Such a structural defect for monooxygenation can be a trade-off for an efficient dioxygenation reaction.…”

“…3), which may be induced by ruffling of hydroxyheme. Further, ruffling of heme has been suggested to promote the initial monooxygenation by changing its electronic structure (16,32). Thus, the unique substrate deformation appears to enhance two different modes of oxygenation at different stages of catalysis to achieve the unprecedented dual function of MhuD.…”

“…The Raman shifts were calibrated with indene. Oxy-ferrous heme-MhuD in 0.1 M potassium phosphate, pH 7.0 was prepared by adding either 16 O 2 or 18 O 2 to ferrous heme-MhuD just before the measurements. The ferrous heme-MhuD solution was prepared in the anaerobic glove box by reducing the ferric heme-MhuD with excess sodium dithionite, which was removed by gel filtration chromatography using a G-25 column.…”

“…1C and Fig. S1) (15,16). The ruffling is expected to drastically modulate the hemedependent O 2 activation to initiate this unique mechanism (14).…”

Time-resolved Studies of IsdG Protein Identify Molecular Signposts along the Non-canonical Heme Oxygenase Pathway

“…Instead of biliverdin IX␣ and CO, the IsdG protein from Mycobacterium tuberculosis (known as MhuD) generates triply-oxygenated linear tetrapyrroles called mycobilins ( Fig. 1) (14,15). A formyl group remains appended to pyrrole ring A or B at the site of macrocycle cleavage, and an oxo group is generated on the pyrrole ring on the opposite side.…”