Solution NMR study of environmental effects on substrate seating in human heme oxygenase: Influence of polypeptide truncation, substrate modification and axial ligand

Zhu, Wenfeng; Li, Yiming; Wang, Jinling; Montellano, Paul R. Ortiz de; Mar, Gerd N. La

doi:10.1016/j.jinorgbio.2005.08.010

Cited by 11 publications

(24 citation statements)

References 47 publications

(174 reference statements)

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“…Systematic changes in active site dipolar shift pattern for a largely conserved molecular structure infer changes in the orientation of χ, which can be indirectly related to small systematic structural changes in the active site (15, 22, 31, 44). …”

Section: Resultsmentioning

confidence: 99%

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Influence of Substrate Modification and C-Terminal Truncation on the Active Site Structure of Substrate-Bound Heme Oxygenase from Neisseriae meningitidis. A ¹H NMR Study

Peng

Satterlee

et al. 2011

Biochemistry

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Heme oxygenase, HO, from the pathogenic bacterium N. meningitidis, NmHO, which secures host iron, shares many properties with mammalian HOs, but also exhibits some key differences. The crystal structure appears more compact and the crystal-undetected C-terminus interacts with substrate in solution. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D 1H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~102 increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed.

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Section: Resultsmentioning

confidence: 99%

“…For mammalian HOs, the alternate orientations are comparably populated for both native (2V,4V)DH and (2H,4H)DH (20–22). The much stronger selectivity between the two substrate orientations in Nm HO arises from increased hydrophobic interactions between the substrate and the protein matrix, in particular at the pyrrole I/II interface.…”

Section: Discussionmentioning

confidence: 99%

Influence of Substrate Modification and C-Terminal Truncation on the Active Site Structure of Substrate-Bound Heme Oxygenase from Neisseriae meningitidis. A ¹H NMR Study

Peng

Satterlee

et al. 2011

Biochemistry

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“…These product regioisomer differences are not surprising for three reasons. First, it is known that the equilibrium protoheme seating preference in the wild-type about the α-γ-meso axis differs significantly in the cyanide relative to the aquo complexes (62), such that the equilibrium isomer distribution for the cyanide complex will not necessarily correspond to that for the resting state aquo complex, nor to that for the activated hydroperoxy species. Second, the "rotated" position of the protohemin in the different isomers of the mutant may not correspond precisely to a 90° difference, so that not all heme seatings may place a meso-position in the "most reactive" position relative to the blockage of three meso-Hs and the steric directing of the Fe-OOH toward the fourth meso position.…”

Section: Discussionmentioning

confidence: 99%

Alteration of the Regiospecificity of Human Heme Oxygenase-1 by Unseating of the Heme but Not Disruption of the Distal Hydrogen Bonding Network

et al. 2005

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Heme oxygenase regiospecifically oxidizes heme at the α-meso position to give biliverdin IXα, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry, but partially shifts the oxidation to the β/δ-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by ~90°, causes a slight loss of regiospecificity, but combined with the R183E and K18E mutations results primarily in β/δ-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/ E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network, impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane.

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“…Instead we employ the two-fold symmetric heme, 2,4-dimethyldeuterohemin, DMDH (R = CH 3 in Figure 2A), which was found (18) to generate a single, homogeneous species in solution with a molecular structure essentially indistinguishable from that of the PH complex. (20, 47, 48) To assess whether any NMR spectroscopic anomalies are related to the distinctive HO distal pocket environment that stabilizes hemolytic over heterolytic O-O bond scission, we include 1 H NMR data on the Asp140Ala point mutant, D140A-hHO-PH-N 3 , which abolishes mammalian HO activity, (32, 33) and instead favors heterolytic O-O bond cleavage.…”

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The Orbital Ground State of the Azide−Substrate Complex of Human Heme Oxygenase Is an Indicator of Distal H-Bonding: Implications for the Enzyme Mechanism

et al. 2009

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The active site electronic structure of the azide complex of substrate-bound human heme oxygenase-1, (hHO) has been investigated by 1H NMR spectroscopy to shed light on the orbital/spin ground state as an indicator of the unique distal pocket environment of the enzyme. 2D 1H NMR assignments of the substrate and substrate-contact residue signals reveal a pattern of substrate methyl contact shifts, that places the lone iron π-spin in the dxz orbital, rather than the dyz orbital found in the cyanide complex. Comparison of iron spin relaxivity, magnetic anisotropy and magnetic susceptibilities argues for a low-spin, (dxy)2(dyz,dxz)3, ground state in both azide and cyanide complexes. The switch from singly-occupied dyz for the cyanide to dxz for the azide complex of hHO is shown to be consistent with the orbital hole determined by the azide π-plane in the latter complex, which is ∼90° in-plane rotated from that of the imidazole π-plane. The induction of the altered orbital ground state in the azide relative to the cyanide hHO complex, as well as the mean low-field bias of methyl hyperfine shifts and their paramagnetic relaxivity relative to those in globins, indicate that azide exerts a stronger ligand field in hHO than in the globins, or that the distal H-bonding to azide is weaker in hHO than in globins. The Asp140 → Ala hHO mutant that abolishes activity retains the unusual WT azide complex spin/orbital ground state. The relevance of our findings for other HO complexes and the HO mechanism is discussed.

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Solution NMR study of environmental effects on substrate seating in human heme oxygenase: Influence of polypeptide truncation, substrate modification and axial ligand

Cited by 11 publications

References 47 publications

Influence of Substrate Modification and C-Terminal Truncation on the Active Site Structure of Substrate-Bound Heme Oxygenase from Neisseriae meningitidis. A ¹H NMR Study

Influence of Substrate Modification and C-Terminal Truncation on the Active Site Structure of Substrate-Bound Heme Oxygenase from Neisseriae meningitidis. A ¹H NMR Study

Alteration of the Regiospecificity of Human Heme Oxygenase-1 by Unseating of the Heme but Not Disruption of the Distal Hydrogen Bonding Network

The Orbital Ground State of the Azide−Substrate Complex of Human Heme Oxygenase Is an Indicator of Distal H-Bonding: Implications for the Enzyme Mechanism

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Solution NMR study of environmental effects on substrate seating in human heme oxygenase: Influence of polypeptide truncation, substrate modification and axial ligand

Cited by 11 publications

References 47 publications

Influence of Substrate Modification and C-Terminal Truncation on the Active Site Structure of Substrate-Bound Heme Oxygenase from Neisseriae meningitidis. A 1H NMR Study

Influence of Substrate Modification and C-Terminal Truncation on the Active Site Structure of Substrate-Bound Heme Oxygenase from Neisseriae meningitidis. A 1H NMR Study

Alteration of the Regiospecificity of Human Heme Oxygenase-1 by Unseating of the Heme but Not Disruption of the Distal Hydrogen Bonding Network

The Orbital Ground State of the Azide−Substrate Complex of Human Heme Oxygenase Is an Indicator of Distal H-Bonding: Implications for the Enzyme Mechanism

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Influence of Substrate Modification and C-Terminal Truncation on the Active Site Structure of Substrate-Bound Heme Oxygenase from Neisseriae meningitidis. A ¹H NMR Study

Influence of Substrate Modification and C-Terminal Truncation on the Active Site Structure of Substrate-Bound Heme Oxygenase from Neisseriae meningitidis. A ¹H NMR Study