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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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VTVH MCD saturation magnetization curves recorded at 2, 5, and10 K for MhuD–heme–CN along with simulated saturationmagnetization curves for two low-spin Fe(III) model complexes.43 The VTVH MCD data strongly suggests that theelectronic ground state of MhuD–heme–CN is 2B2g and that there exists a low-lying 2Eg electronic excited state.
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fig7: VTVH MCD saturation magnetization curves recorded at 2, 5, and10 K for MhuD–heme–CN along with simulated saturationmagnetization curves for two low-spin Fe(III) model complexes.43 The VTVH MCD data strongly suggests that theelectronic ground state of MhuD–heme–CN is 2B2g and that there exists a low-lying 2Eg electronic excited state.

Mentions: In order to distinguish between 2Eg and 2B2g electronicground states, we measured VTVH MCD saturation curves for the Soretband trough (424 nm) of MhuD–heme–CN at 2, 5, and 10K (Figure 7). The curves for MhuD–heme–CNclosely align with an S = 1/2 iron center.42 However, there is somenesting of the saturation magnetization curves. Nesting of saturationmagnetization curves is typically associated with either a high-spinsystem or coupled-spin systems. On the basis of the π* acceptorstrength of a cyanide ligand, it is extremely unlikely that an Fe(III)–CNcomplex would form a high-spin species. Indeed, the MhuD–heme–CNcurves do not align with those of a high-spin complex (Figure S7). If the nesting of the MhuD–heme–CNsaturation magnetization arises from a coupled spin system, then itwould most likely be a small fraction of MhuD–diheme–CN.The MhuD–diheme–CN curves are less nested than the MhuD–heme–CNcurves, indicating that a MhuD–diheme–CN fraction isnot the source of nesting in the MhuD–heme–CN data (Figure S8). Although nesting is unusual for anisolated S = 1/2 center, itis possible if a thermal mixture of two or more S = 1/2 states are populated. On the basis ofthe NMR and MCD spectroscopic data presented up to this point, thisis a realistic possibility for MhuD–heme–CN. To investigatethis possibility further, we simulated VTVH MCD saturation magnetizationcurves for two Fe(III) porphyrin model complexes, one with a 2Eg electronic ground state andthe other with a 2B2g groundstate.


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)

VTVH MCD saturation magnetization curves recorded at 2, 5, and10 K for MhuD–heme–CN along with simulated saturationmagnetization curves for two low-spin Fe(III) model complexes.43 The VTVH MCD data strongly suggests that theelectronic ground state of MhuD–heme–CN is 2B2g and that there exists a low-lying 2Eg electronic excited state.
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Related In: Results  -  Collection

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

fig7: VTVH MCD saturation magnetization curves recorded at 2, 5, and10 K for MhuD–heme–CN along with simulated saturationmagnetization curves for two low-spin Fe(III) model complexes.43 The VTVH MCD data strongly suggests that theelectronic ground state of MhuD–heme–CN is 2B2g and that there exists a low-lying 2Eg electronic excited state.
Mentions: In order to distinguish between 2Eg and 2B2g electronicground states, we measured VTVH MCD saturation curves for the Soretband trough (424 nm) of MhuD–heme–CN at 2, 5, and 10K (Figure 7). The curves for MhuD–heme–CNclosely align with an S = 1/2 iron center.42 However, there is somenesting of the saturation magnetization curves. Nesting of saturationmagnetization curves is typically associated with either a high-spinsystem or coupled-spin systems. On the basis of the π* acceptorstrength of a cyanide ligand, it is extremely unlikely that an Fe(III)–CNcomplex would form a high-spin species. Indeed, the MhuD–heme–CNcurves do not align with those of a high-spin complex (Figure S7). If the nesting of the MhuD–heme–CNsaturation magnetization arises from a coupled spin system, then itwould most likely be a small fraction of MhuD–diheme–CN.The MhuD–diheme–CN curves are less nested than the MhuD–heme–CNcurves, indicating that a MhuD–diheme–CN fraction isnot the source of nesting in the MhuD–heme–CN data (Figure S8). Although nesting is unusual for anisolated S = 1/2 center, itis possible if a thermal mixture of two or more S = 1/2 states are populated. On the basis ofthe NMR and MCD spectroscopic data presented up to this point, thisis a realistic possibility for MhuD–heme–CN. To investigatethis possibility further, we simulated VTVH MCD saturation magnetizationcurves for two Fe(III) porphyrin model complexes, one with a 2Eg electronic ground state andthe other with a 2B2g groundstate.

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