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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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Structural comparison of the active sites ofMhuD–heme–CNand MhuD–diheme. (A) Superposition of MhuD–heme–CN(PDB ID 4NL5, cyan) with MhuD–diheme (PDB ID 3HX9, pink) shows that α2 in MhuD–heme–CNis kinked, whereas it is extended in the MhuD–diheme structure.This kink results in MhuD–heme–CN His75 moving 4.5 Åto coordinate heme iron (orange spheres). (B, C) The orientationsof most residues within the MhuD–heme–CN (B) and MhuD–diheme(C) active sites are unchanged, but Arg26 in the MhuD–heme–CNstructure flips into the reduced volume active site. Heme propionates6 and 7 are denoted by P6 and P7, respectively, and the solvent-exposedheme propionates (MhuD–diheme) are differentiated with a primesymbol (′). Heme molecules are in stick representation, withcarbon atoms depicted in light cyan and light pink for MhuD–heme–CNand MhuD–diheme, respectively, and nitrogen, oxygen, and cyanocarbon atoms in blue, red, and yellow, respectively. Iron atoms, anordered Cl– atom (MhuD–diheme structure),and water molecule (W1, MhuD–heme–CN structure) aredepicted as orange, green, and red spheres, respectively.
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fig3: Structural comparison of the active sites ofMhuD–heme–CNand MhuD–diheme. (A) Superposition of MhuD–heme–CN(PDB ID 4NL5, cyan) with MhuD–diheme (PDB ID 3HX9, pink) shows that α2 in MhuD–heme–CNis kinked, whereas it is extended in the MhuD–diheme structure.This kink results in MhuD–heme–CN His75 moving 4.5 Åto coordinate heme iron (orange spheres). (B, C) The orientationsof most residues within the MhuD–heme–CN (B) and MhuD–diheme(C) active sites are unchanged, but Arg26 in the MhuD–heme–CNstructure flips into the reduced volume active site. Heme propionates6 and 7 are denoted by P6 and P7, respectively, and the solvent-exposedheme propionates (MhuD–diheme) are differentiated with a primesymbol (′). Heme molecules are in stick representation, withcarbon atoms depicted in light cyan and light pink for MhuD–heme–CNand MhuD–diheme, respectively, and nitrogen, oxygen, and cyanocarbon atoms in blue, red, and yellow, respectively. Iron atoms, anordered Cl– atom (MhuD–diheme structure),and water molecule (W1, MhuD–heme–CN structure) aredepicted as orange, green, and red spheres, respectively.

Mentions: The MhuD–dihemeand MhuD–heme–CN structuressuperimpose with a root-mean-square deviation (rmsd) of 0.29 Åover all Cα atoms.5 Within the activesite pocket, the heme substrate overlays with the solvent-protectedheme from the MhuD–diheme structure; however, the modeled hemeis rotated 180° about the α–γ axis (Figure 3A). Additionally, there is an increase in heme out-of-planedistortion. The distortions of the hemes in MhuD–heme–CN(1.4 and 1.5 Å) are more severe than those in the solvent-protectedheme from MhuD–diheme (0.7 Å), as analyzed by normal-coordinatestructural decomposition.45 It was suggestedthat Phe22 contributes to heme ruffling, the b1u component of the heme out-of-plane distortion, in IsdG bycontacting the γ-meso carbon.19,31 In the MhuD–dihemeand MhuD–heme–CN structures, the side chains of thecorresponding residue, Phe23, overlay, suggesting that Phe23 doesnot contribute to ruffling, as the MhuD–diheme solvent-protectedheme is planar compared to the distorted MhuD–heme–CNporphyrin ring (Figure 3A). However, the MhuD–dihemesecond solvent-exposed heme may play a role in the planar nature ofthe solvent-protected heme. The most notable structural differencesare within the α2 helix and the subsequent loop region surroundingthe active site (Figure 3A). In MhuD–heme–CN,the α2 helix is kinked after residue Asn68, whereas in the MhuD–dihemestructure, this helix (α2) is extended. This kink results inthe movement of His75 (4.5 Å) so that it may coordinate withheme iron in the MhuD–heme–CN structure (Figure 3B). Furthermore, there is one notable altered sidechain conformation, Arg26, between the MhuD–heme–CNand MhuD–diheme structures. In the MhuD–diheme structure,Arg26 forms an H-bond with both propionate 6 (P6′) and propionate7 (P7′) from the solvent-exposed heme but not with the solvent-protectedheme molecule (Figure 3C). However, in MhuD–heme–CN,Arg26 is flipped into the reduced volume heme active site, where itH-bonds to an active site water molecule (W1), which in turn H-bondsto the carbonyl oxygen of His75 and heme propionate 7 (P7), whoseorientation is rotated toward the active site water (W1, Figure 3B). Arg26 is also in H-bonding distance to hemepropionate 6 (P6). One could speculate that the ordered water andalternative conformation of the Arg26 side chain may stabilize theotherwise flexible loop to form a stable monoheme active site. Theposition of this water molecule is conserved in both active sitesof the MhuD dimer; however, in one of two active sites of the dimer,there is a second water molecule that also H-bonds with Arg26 (notshown).


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)

Structural comparison of the active sites ofMhuD–heme–CNand MhuD–diheme. (A) Superposition of MhuD–heme–CN(PDB ID 4NL5, cyan) with MhuD–diheme (PDB ID 3HX9, pink) shows that α2 in MhuD–heme–CNis kinked, whereas it is extended in the MhuD–diheme structure.This kink results in MhuD–heme–CN His75 moving 4.5 Åto coordinate heme iron (orange spheres). (B, C) The orientationsof most residues within the MhuD–heme–CN (B) and MhuD–diheme(C) active sites are unchanged, but Arg26 in the MhuD–heme–CNstructure flips into the reduced volume active site. Heme propionates6 and 7 are denoted by P6 and P7, respectively, and the solvent-exposedheme propionates (MhuD–diheme) are differentiated with a primesymbol (′). Heme molecules are in stick representation, withcarbon atoms depicted in light cyan and light pink for MhuD–heme–CNand MhuD–diheme, respectively, and nitrogen, oxygen, and cyanocarbon atoms in blue, red, and yellow, respectively. Iron atoms, anordered Cl– atom (MhuD–diheme structure),and water molecule (W1, MhuD–heme–CN structure) aredepicted as orange, green, and red spheres, respectively.
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fig3: Structural comparison of the active sites ofMhuD–heme–CNand MhuD–diheme. (A) Superposition of MhuD–heme–CN(PDB ID 4NL5, cyan) with MhuD–diheme (PDB ID 3HX9, pink) shows that α2 in MhuD–heme–CNis kinked, whereas it is extended in the MhuD–diheme structure.This kink results in MhuD–heme–CN His75 moving 4.5 Åto coordinate heme iron (orange spheres). (B, C) The orientationsof most residues within the MhuD–heme–CN (B) and MhuD–diheme(C) active sites are unchanged, but Arg26 in the MhuD–heme–CNstructure flips into the reduced volume active site. Heme propionates6 and 7 are denoted by P6 and P7, respectively, and the solvent-exposedheme propionates (MhuD–diheme) are differentiated with a primesymbol (′). Heme molecules are in stick representation, withcarbon atoms depicted in light cyan and light pink for MhuD–heme–CNand MhuD–diheme, respectively, and nitrogen, oxygen, and cyanocarbon atoms in blue, red, and yellow, respectively. Iron atoms, anordered Cl– atom (MhuD–diheme structure),and water molecule (W1, MhuD–heme–CN structure) aredepicted as orange, green, and red spheres, respectively.
Mentions: The MhuD–dihemeand MhuD–heme–CN structuressuperimpose with a root-mean-square deviation (rmsd) of 0.29 Åover all Cα atoms.5 Within the activesite pocket, the heme substrate overlays with the solvent-protectedheme from the MhuD–diheme structure; however, the modeled hemeis rotated 180° about the α–γ axis (Figure 3A). Additionally, there is an increase in heme out-of-planedistortion. The distortions of the hemes in MhuD–heme–CN(1.4 and 1.5 Å) are more severe than those in the solvent-protectedheme from MhuD–diheme (0.7 Å), as analyzed by normal-coordinatestructural decomposition.45 It was suggestedthat Phe22 contributes to heme ruffling, the b1u component of the heme out-of-plane distortion, in IsdG bycontacting the γ-meso carbon.19,31 In the MhuD–dihemeand MhuD–heme–CN structures, the side chains of thecorresponding residue, Phe23, overlay, suggesting that Phe23 doesnot contribute to ruffling, as the MhuD–diheme solvent-protectedheme is planar compared to the distorted MhuD–heme–CNporphyrin ring (Figure 3A). However, the MhuD–dihemesecond solvent-exposed heme may play a role in the planar nature ofthe solvent-protected heme. The most notable structural differencesare within the α2 helix and the subsequent loop region surroundingthe active site (Figure 3A). In MhuD–heme–CN,the α2 helix is kinked after residue Asn68, whereas in the MhuD–dihemestructure, this helix (α2) is extended. This kink results inthe movement of His75 (4.5 Å) so that it may coordinate withheme iron in the MhuD–heme–CN structure (Figure 3B). Furthermore, there is one notable altered sidechain conformation, Arg26, between the MhuD–heme–CNand MhuD–diheme structures. In the MhuD–diheme structure,Arg26 forms an H-bond with both propionate 6 (P6′) and propionate7 (P7′) from the solvent-exposed heme but not with the solvent-protectedheme molecule (Figure 3C). However, in MhuD–heme–CN,Arg26 is flipped into the reduced volume heme active site, where itH-bonds to an active site water molecule (W1), which in turn H-bondsto the carbonyl oxygen of His75 and heme propionate 7 (P7), whoseorientation is rotated toward the active site water (W1, Figure 3B). Arg26 is also in H-bonding distance to hemepropionate 6 (P6). One could speculate that the ordered water andalternative conformation of the Arg26 side chain may stabilize theotherwise flexible loop to form a stable monoheme active site. Theposition of this water molecule is conserved in both active sitesof the MhuD dimer; however, in one of two active sites of the dimer,there is a second water molecule that also H-bonds with Arg26 (notshown).

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