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Crystallographic and spectroscopic insights into heme degradation by Mycobacterium tuberculosis MhuD.

Graves AB, Morse RP, Chao A, Iniguez A, Goulding CW, Liptak MD - Inorg Chem (2014)

Bottom Line: 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.Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Vermont , Burlington, Vermont 05405, United States.

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 (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 3dxz-, 3dyz-, and 3dxy-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)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g 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 (2)B2g electronic ground state with a low-lying (2)Eg 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.

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X-ray crystal structure of MhuD–heme–CN(PDB ID 4NL5). (A) Ribbon representationof the dimeric MhuD–heme–CN complex. (B) Ribbon representationof the MhuD–heme–CN heme-binding pocket. α-Helicesand β-strands are depicted in cyan and magenta, respectively.Loops and side chain carbons are shown in salmon. All α-helicesare labeled, with the second polypeptide chain differentiated by aprime symbol (′). Heme–CN, one per active site, is representedas a stick model, where nitrogen, oxygen, heme carbon, and cyano carbonatoms are in blue, red, white, and yellow, respectively. Iron atomsand ordered water molecules are depicted as orange and red spheres,respectively.
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fig2: X-ray crystal structure of MhuD–heme–CN(PDB ID 4NL5). (A) Ribbon representationof the dimeric MhuD–heme–CN complex. (B) Ribbon representationof the MhuD–heme–CN heme-binding pocket. α-Helicesand β-strands are depicted in cyan and magenta, respectively.Loops and side chain carbons are shown in salmon. All α-helicesare labeled, with the second polypeptide chain differentiated by aprime symbol (′). Heme–CN, one per active site, is representedas a stick model, where nitrogen, oxygen, heme carbon, and cyano carbonatoms are in blue, red, white, and yellow, respectively. Iron atomsand ordered water molecules are depicted as orange and red spheres,respectively.

Mentions: The crystal structure of MhuD–dihemewas previously determined, which revealed two stacked heme moleculesin the MhuD active site. However, no heme-degradation activity wasobserved by MhuD–diheme, and only the monoheme complex is capableof degrading heme.5 To observe MhuD inits monoheme active conformation, we determined the structure of MhuD–heme–CNto 1.9 Å resolution, with one homodimer in the asymmetric unit.MhuD–heme–CN retains its ferrodoxin-like α + β-barrelfold, as observed for MhuD–diheme;5 however, only one bound molecule of heme is observed in each activesite (Figure 2A). In accord with previouslyreported spectroscopic data,6 His75 coordinatesthe iron of MhuD–heme–CN on its proximal side (2.1 Å,Figure 2B), and the His75 imidazole ring ishydrogen (H)-bonded to the backbone carbonyl of Ala71. A fully occupiedcyano group was modeled into the electron density observed on thedistal side of the heme iron, with an Fe–C bond length of 2.1Å. The bound CN atoms refine with B-factors of approximately22 Å2, similar to those of the heme irons, and fitthe electron density well (Figure S5).The CN ligands are observed in a bent coordinating mode, with Fe–C–Nangles of 118 and 120° for chains A and B, respectively, whereasthe Fe–C–N bonds are nearly perpendicular to the porphyrinplane in the IsdI–heme–CN structure, with Fe–C–Nangles of 171° and 158°.30 TheFe–C–N angle observed for MhuD–heme–CNis more similar to the 139° angle seen in cyanide-inhibited ratheme oxygenase (rHO–heme–CN; PDB ID 2E7E) at pH 6.8.44 In the MhuD–heme–CN active site,the CN ligand forms an H-bond with Asn7 NH1 and points toward pyrrolering A, which separates the γ- and δ-meso carbons. Furthermore,the CN-inhibited heme substrate is stabilized by hydrophobic interactionswith Ile9, Phe23, Phe39, Val53, Thr55, Phe63, and Trp66; H-bonds betweenpropionate 6 and Arg22 NH1, Arg26 NH2, and the Val83 backbone amide;and H-bonds between the bent propionate 7 and a water molecule (W1),which in turn H-bonds to Arg26 NH1.


Crystallographic and spectroscopic insights into heme degradation by Mycobacterium tuberculosis MhuD.

Graves AB, Morse RP, Chao A, Iniguez A, Goulding CW, Liptak MD - Inorg Chem (2014)

X-ray crystal structure of MhuD–heme–CN(PDB ID 4NL5). (A) Ribbon representationof the dimeric MhuD–heme–CN complex. (B) Ribbon representationof the MhuD–heme–CN heme-binding pocket. α-Helicesand β-strands are depicted in cyan and magenta, respectively.Loops and side chain carbons are shown in salmon. All α-helicesare labeled, with the second polypeptide chain differentiated by aprime symbol (′). Heme–CN, one per active site, is representedas a stick model, where nitrogen, oxygen, heme carbon, and cyano carbonatoms are in blue, red, white, and yellow, respectively. Iron atomsand ordered water molecules are depicted as orange and red spheres,respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4060609&req=5

fig2: X-ray crystal structure of MhuD–heme–CN(PDB ID 4NL5). (A) Ribbon representationof the dimeric MhuD–heme–CN complex. (B) Ribbon representationof the MhuD–heme–CN heme-binding pocket. α-Helicesand β-strands are depicted in cyan and magenta, respectively.Loops and side chain carbons are shown in salmon. All α-helicesare labeled, with the second polypeptide chain differentiated by aprime symbol (′). Heme–CN, one per active site, is representedas a stick model, where nitrogen, oxygen, heme carbon, and cyano carbonatoms are in blue, red, white, and yellow, respectively. Iron atomsand ordered water molecules are depicted as orange and red spheres,respectively.
Mentions: The crystal structure of MhuD–dihemewas previously determined, which revealed two stacked heme moleculesin the MhuD active site. However, no heme-degradation activity wasobserved by MhuD–diheme, and only the monoheme complex is capableof degrading heme.5 To observe MhuD inits monoheme active conformation, we determined the structure of MhuD–heme–CNto 1.9 Å resolution, with one homodimer in the asymmetric unit.MhuD–heme–CN retains its ferrodoxin-like α + β-barrelfold, as observed for MhuD–diheme;5 however, only one bound molecule of heme is observed in each activesite (Figure 2A). In accord with previouslyreported spectroscopic data,6 His75 coordinatesthe iron of MhuD–heme–CN on its proximal side (2.1 Å,Figure 2B), and the His75 imidazole ring ishydrogen (H)-bonded to the backbone carbonyl of Ala71. A fully occupiedcyano group was modeled into the electron density observed on thedistal side of the heme iron, with an Fe–C bond length of 2.1Å. The bound CN atoms refine with B-factors of approximately22 Å2, similar to those of the heme irons, and fitthe electron density well (Figure S5).The CN ligands are observed in a bent coordinating mode, with Fe–C–Nangles of 118 and 120° for chains A and B, respectively, whereasthe Fe–C–N bonds are nearly perpendicular to the porphyrinplane in the IsdI–heme–CN structure, with Fe–C–Nangles of 171° and 158°.30 TheFe–C–N angle observed for MhuD–heme–CNis more similar to the 139° angle seen in cyanide-inhibited ratheme oxygenase (rHO–heme–CN; PDB ID 2E7E) at pH 6.8.44 In the MhuD–heme–CN active site,the CN ligand forms an H-bond with Asn7 NH1 and points toward pyrrolering A, which separates the γ- and δ-meso carbons. Furthermore,the CN-inhibited heme substrate is stabilized by hydrophobic interactionswith Ile9, Phe23, Phe39, Val53, Thr55, Phe63, and Trp66; H-bonds betweenpropionate 6 and Arg22 NH1, Arg26 NH2, and the Val83 backbone amide;and H-bonds between the bent propionate 7 and a water molecule (W1),which in turn H-bonds to Arg26 NH1.

Bottom Line: 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.Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Vermont , Burlington, Vermont 05405, United States.

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 (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 3dxz-, 3dyz-, and 3dxy-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)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g 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 (2)B2g electronic ground state with a low-lying (2)Eg 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.

Show MeSH
Related in: MedlinePlus