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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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HO has an ordered network of active site watermolecules on thedistal side of the heme substrate and is believed to stabilize a 2Eg electronic state, where spindensity is delocalized onto the porphyrin pyrrole rings (violet andblue circles represent the two components of this degenerate state),producing biliverdin, CO, and iron as products. S.aureus IsdI has been proposed to stabilize a 2B2g state, with spin density delocalizedonto the α-, β-, γ-, and δ-meso carbons (greencircles), without a defined network of water molecules en route tostaphylobilin and formaldehyde formation. M. tuberculosis MhuD degrades heme to mycobilin and iron, but prior to this work,the active site and electronic structures were unknown.
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fig1: HO has an ordered network of active site watermolecules on thedistal side of the heme substrate and is believed to stabilize a 2Eg electronic state, where spindensity is delocalized onto the porphyrin pyrrole rings (violet andblue circles represent the two components of this degenerate state),producing biliverdin, CO, and iron as products. S.aureus IsdI has been proposed to stabilize a 2B2g state, with spin density delocalizedonto the α-, β-, γ-, and δ-meso carbons (greencircles), without a defined network of water molecules en route tostaphylobilin and formaldehyde formation. M. tuberculosis MhuD degrades heme to mycobilin and iron, but prior to this work,the active site and electronic structures were unknown.

Mentions: Although MhuD degradesheme to non-heme iron and organic byproducts,the enzyme has features that distinguish it from canonical heme oxygenases(HOs).5,6 The truncated, soluble forms of human andrat HO adopt α-helical folds.9−11 HO enzymes from severalpathogenic bacteria also possess α-helical folds, including Neisseria meningitides HemO,12Corynebacterium diphtheria HmuO,13 and Pseudomonas aeruginosa PigA.14 In contrast, the catalyticallyinactive, diheme-bound form of MhuD (MhuD–diheme) has a ferrodoxin-likefold similar to that of the IsdG and IsdI heme-degrading enzymes from Staphylococcus aureus.5,15 In addition,the organic byproducts of MhuD-catalyzed heme degradation are unique(Figure 1). Most canonical HOs convert theporphyrin ring to α-biliverdin and carbon monoxide (CO).16P. aeruginosa PigA is an exception, where rotation of the heme substrate withinthe enzyme active site results in formation of β- and δ-biliverdin.17,18 On the other hand, S. aureus IsdGand IsdI convert the porphyrin ring to a mixture of the β- andδ-isomers of staphylobilin along with a molecule of formaldehyde.19,20M. tuberculosis MhuD generates aproduct distinct from those of canonical HOs or IsdG/I, mycobilin,where the porphyrin ring of heme is cleaved at the α-meso carbon,and this carbon is retained as an aldehyde group.6 Because MhuD generates different enzymatic products ascompared to those of HOs, IsdG, or IsdI, the reaction must proceedvia a unique mechanism.


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)

HO has an ordered network of active site watermolecules on thedistal side of the heme substrate and is believed to stabilize a 2Eg electronic state, where spindensity is delocalized onto the porphyrin pyrrole rings (violet andblue circles represent the two components of this degenerate state),producing biliverdin, CO, and iron as products. S.aureus IsdI has been proposed to stabilize a 2B2g state, with spin density delocalizedonto the α-, β-, γ-, and δ-meso carbons (greencircles), without a defined network of water molecules en route tostaphylobilin and formaldehyde formation. M. tuberculosis MhuD degrades heme to mycobilin and iron, but prior to this work,the active site and electronic structures were unknown.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: HO has an ordered network of active site watermolecules on thedistal side of the heme substrate and is believed to stabilize a 2Eg electronic state, where spindensity is delocalized onto the porphyrin pyrrole rings (violet andblue circles represent the two components of this degenerate state),producing biliverdin, CO, and iron as products. S.aureus IsdI has been proposed to stabilize a 2B2g state, with spin density delocalizedonto the α-, β-, γ-, and δ-meso carbons (greencircles), without a defined network of water molecules en route tostaphylobilin and formaldehyde formation. M. tuberculosis MhuD degrades heme to mycobilin and iron, but prior to this work,the active site and electronic structures were unknown.
Mentions: Although MhuD degradesheme to non-heme iron and organic byproducts,the enzyme has features that distinguish it from canonical heme oxygenases(HOs).5,6 The truncated, soluble forms of human andrat HO adopt α-helical folds.9−11 HO enzymes from severalpathogenic bacteria also possess α-helical folds, including Neisseria meningitides HemO,12Corynebacterium diphtheria HmuO,13 and Pseudomonas aeruginosa PigA.14 In contrast, the catalyticallyinactive, diheme-bound form of MhuD (MhuD–diheme) has a ferrodoxin-likefold similar to that of the IsdG and IsdI heme-degrading enzymes from Staphylococcus aureus.5,15 In addition,the organic byproducts of MhuD-catalyzed heme degradation are unique(Figure 1). Most canonical HOs convert theporphyrin ring to α-biliverdin and carbon monoxide (CO).16P. aeruginosa PigA is an exception, where rotation of the heme substrate withinthe enzyme active site results in formation of β- and δ-biliverdin.17,18 On the other hand, S. aureus IsdGand IsdI convert the porphyrin ring to a mixture of the β- andδ-isomers of staphylobilin along with a molecule of formaldehyde.19,20M. tuberculosis MhuD generates aproduct distinct from those of canonical HOs or IsdG/I, mycobilin,where the porphyrin ring of heme is cleaved at the α-meso carbon,and this carbon is retained as an aldehyde group.6 Because MhuD generates different enzymatic products ascompared to those of HOs, IsdG, or IsdI, the reaction must proceedvia a unique mechanism.

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