Crystallographic and Spectroscopic Insights into Heme Degradation by Mycobacterium tuberculosis

[Image: see text] 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 (1)H 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 3d(xz)-, 3d(yz)-, and 3d(xy)-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD–heme–CN is consistent with population of both a (2)E(g) electronic state with a (d(xy))(2)(d(xz),d(yz))(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B(2g) state with a (d(xz),d(yz))(4)(d(xy))(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 (2)B(2g) electronic ground state with a low-lying (2)E(g) 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

Identification of an Escherichia coli O157:H7 heme oxygenase with tandem functional repeats

Heme oxygenases (HOs) catalyze the oxidation of heme to biliverdin, carbon monoxide (CO), and free iron. Iron acquisition is critical for invading microorganisms to enable survival and growth. Here we report the crystal structure of ChuS, which displays a previously uncharacterized fold and is unique compared with other characterized HOs. Despite only 19% sequence identity between the N- and C-terminal halves, these segments of ChuS represent a structural duplication, with a root-mean-square deviation of 2.1 Å between the two repeats. ChuS is capable of using ascorbic acid or cytochrome P450 reductase-NADPH as electron sources for heme oxygenation. CO detection confirmed that ChuS is a HO, and we have identified it in pathogenic Escherichia coli O157:H7. Based on sequence analysis, this HO is present in many bacteria, although not in the E. coli K-12 strain. The N- and C-terminal halves of ChuS are each a functional HO