Controlling conformational flexibility of an O 2-binding H-NOX domain

Emily Weinert, Christine M. Phillips-Piro, Rosalie Tran, Richard A. Mathies, Michael A. Marletta

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O 2)-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O 2 association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 Å crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O 2 as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.

Original languageEnglish (US)
Pages (from-to)6832-6840
Number of pages9
JournalBiochemistry
Volume50
Issue number32
DOIs
StatePublished - Aug 16 2011

Fingerprint

Heme
Nitric Oxide
Oxygen
Raman Spectrum Analysis
Phenylalanine
Mutation
Raman spectroscopy
Thermoanaerobacter
Ligands
Isoleucine
X ray crystallography
X Ray Crystallography
Hydrogen Bonding
Scaffolds
Carrier concentration
Conformations
Carrier Proteins
Hydrogen bonds
Proteins
Crystal structure

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Weinert, E., Phillips-Piro, C. M., Tran, R., Mathies, R. A., & Marletta, M. A. (2011). Controlling conformational flexibility of an O 2-binding H-NOX domain. Biochemistry, 50(32), 6832-6840. https://doi.org/10.1021/bi200788x
Weinert, Emily ; Phillips-Piro, Christine M. ; Tran, Rosalie ; Mathies, Richard A. ; Marletta, Michael A. / Controlling conformational flexibility of an O 2-binding H-NOX domain. In: Biochemistry. 2011 ; Vol. 50, No. 32. pp. 6832-6840.
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abstract = "Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O 2)-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O 2 association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 {\AA} crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O 2 as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.",
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Weinert, E, Phillips-Piro, CM, Tran, R, Mathies, RA & Marletta, MA 2011, 'Controlling conformational flexibility of an O 2-binding H-NOX domain', Biochemistry, vol. 50, no. 32, pp. 6832-6840. https://doi.org/10.1021/bi200788x

Controlling conformational flexibility of an O 2-binding H-NOX domain. / Weinert, Emily; Phillips-Piro, Christine M.; Tran, Rosalie; Mathies, Richard A.; Marletta, Michael A.

In: Biochemistry, Vol. 50, No. 32, 16.08.2011, p. 6832-6840.

Research output: Contribution to journalArticle

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T1 - Controlling conformational flexibility of an O 2-binding H-NOX domain

AU - Weinert, Emily

AU - Phillips-Piro, Christine M.

AU - Tran, Rosalie

AU - Mathies, Richard A.

AU - Marletta, Michael A.

PY - 2011/8/16

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N2 - Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O 2)-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O 2 association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 Å crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O 2 as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.

AB - Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O 2)-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O 2 association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 Å crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O 2 as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.

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Weinert E, Phillips-Piro CM, Tran R, Mathies RA, Marletta MA. Controlling conformational flexibility of an O 2-binding H-NOX domain. Biochemistry. 2011 Aug 16;50(32):6832-6840. https://doi.org/10.1021/bi200788x