Correlation between the steric bulk of the distal E7 and E11 residues and the tilt of the FeCN unit in cyanometmyoglobin as determined by NMR from the orientation of the magnetic axes in single and double point mutants

Krishna Rajarathnam, Jun Qin, Gerd N. La Mar, Mark L. Chiu, Stephen G. Sligar

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

The amino acids in the heme pocket of sperm whale myoglobin single E11 and double E7 and E11 point mutants in the metcyano form have been assigned by NMR methods to assess the role of steric bulk in modulating ligand tilt. The five mutants investigated are the single mutants His64(E7)→Gly (H[E7]G), Val68(E11)→Ile (V[E11]I), and Val68(E11)→Ala (V[E11]A) and the double mutants His64-(E7)→Gly:Val68(E11)→Ile (H,V[E7,E11]G,I) and His64(E7)→Gly:Val68(E11)→Ala (H,V[E7,E11]G,A). The dipolar (NOESY) contacts on the proximal side of the heme confirm a conserved molecular structure for all of the mutants. The proximal residue coordinates, together with the dipolar shifts for proximal side residues, quantitatively yield the orientations of the magnetic susceptibility tensors, whose major axis corresponds to the orientation of the ligand. It is observed that upon reduction of the steric bulk in the V[E11]A mutant, the tilt of the ligand is significantly reduced (∼8°) from that in the wild type (WT) (∼16°), with little change in the direction of tilt. In the case of increased steric bulk at position 68 in the V[E11]I mutant, it is observed that the extent and direction of the tilt are essentially the same as in WT, and it is shown that this is due to the fact that Ile68 is oriented in the pocket with its C5H3 directed away from the iron. The removal of the bulky imidazole side chain in the H[E7]G and H[E7]V mutants leaves the extent of tilt unchanged from that in WT, but with a direction of tilt rotated by ∼40° that has been interpreted in terms of the energy surface of the heme pocket [Rajarathnam, K., et al. (1993) Biochemistry 32, 5670-5680]. Hence, the E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand, respectively. In the double mutants, the influences of the E7 and E11 substitutions are essentially additive, with the ligand tilt adopting the direction of the H[E7]G single mutant and the extent of tilt of the V[E11]A or V[E11]I single mutant. The extent of the ligand tilt determined herein for the various cyanometmyoglobin mutants does not correlate with the kinetics or thermodynamics of CO ligation, indicating that factors other than steric effects, such as polarity of the heme pocket, play an important role in modulating ligand binding.

Original languageEnglish (US)
Pages (from-to)5493-5501
Number of pages9
JournalBiochemistry
Volume33
Issue number18
StatePublished - 1994
Externally publishedYes

Fingerprint

Nuclear magnetic resonance
Ligands
Heme
Sperm Whale
Biochemistry
Myoglobin
Carbon Monoxide
cyanometmyoglobin
Molecular Structure
Magnetic susceptibility
Thermodynamics
Interfacial energy
Molecular structure
Tensors
Ligation
Substitution reactions
Iron
Direction compound
Amino Acids
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{b53469aaa7254de9ad2684106789ce88,
title = "Correlation between the steric bulk of the distal E7 and E11 residues and the tilt of the FeCN unit in cyanometmyoglobin as determined by NMR from the orientation of the magnetic axes in single and double point mutants",
abstract = "The amino acids in the heme pocket of sperm whale myoglobin single E11 and double E7 and E11 point mutants in the metcyano form have been assigned by NMR methods to assess the role of steric bulk in modulating ligand tilt. The five mutants investigated are the single mutants His64(E7)→Gly (H[E7]G), Val68(E11)→Ile (V[E11]I), and Val68(E11)→Ala (V[E11]A) and the double mutants His64-(E7)→Gly:Val68(E11)→Ile (H,V[E7,E11]G,I) and His64(E7)→Gly:Val68(E11)→Ala (H,V[E7,E11]G,A). The dipolar (NOESY) contacts on the proximal side of the heme confirm a conserved molecular structure for all of the mutants. The proximal residue coordinates, together with the dipolar shifts for proximal side residues, quantitatively yield the orientations of the magnetic susceptibility tensors, whose major axis corresponds to the orientation of the ligand. It is observed that upon reduction of the steric bulk in the V[E11]A mutant, the tilt of the ligand is significantly reduced (∼8°) from that in the wild type (WT) (∼16°), with little change in the direction of tilt. In the case of increased steric bulk at position 68 in the V[E11]I mutant, it is observed that the extent and direction of the tilt are essentially the same as in WT, and it is shown that this is due to the fact that Ile68 is oriented in the pocket with its C5H3 directed away from the iron. The removal of the bulky imidazole side chain in the H[E7]G and H[E7]V mutants leaves the extent of tilt unchanged from that in WT, but with a direction of tilt rotated by ∼40° that has been interpreted in terms of the energy surface of the heme pocket [Rajarathnam, K., et al. (1993) Biochemistry 32, 5670-5680]. Hence, the E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand, respectively. In the double mutants, the influences of the E7 and E11 substitutions are essentially additive, with the ligand tilt adopting the direction of the H[E7]G single mutant and the extent of tilt of the V[E11]A or V[E11]I single mutant. The extent of the ligand tilt determined herein for the various cyanometmyoglobin mutants does not correlate with the kinetics or thermodynamics of CO ligation, indicating that factors other than steric effects, such as polarity of the heme pocket, play an important role in modulating ligand binding.",
author = "Krishna Rajarathnam and Jun Qin and {La Mar}, {Gerd N.} and Chiu, {Mark L.} and Sligar, {Stephen G.}",
year = "1994",
language = "English (US)",
volume = "33",
pages = "5493--5501",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "18",

}

TY - JOUR

T1 - Correlation between the steric bulk of the distal E7 and E11 residues and the tilt of the FeCN unit in cyanometmyoglobin as determined by NMR from the orientation of the magnetic axes in single and double point mutants

AU - Rajarathnam, Krishna

AU - Qin, Jun

AU - La Mar, Gerd N.

AU - Chiu, Mark L.

AU - Sligar, Stephen G.

PY - 1994

Y1 - 1994

N2 - The amino acids in the heme pocket of sperm whale myoglobin single E11 and double E7 and E11 point mutants in the metcyano form have been assigned by NMR methods to assess the role of steric bulk in modulating ligand tilt. The five mutants investigated are the single mutants His64(E7)→Gly (H[E7]G), Val68(E11)→Ile (V[E11]I), and Val68(E11)→Ala (V[E11]A) and the double mutants His64-(E7)→Gly:Val68(E11)→Ile (H,V[E7,E11]G,I) and His64(E7)→Gly:Val68(E11)→Ala (H,V[E7,E11]G,A). The dipolar (NOESY) contacts on the proximal side of the heme confirm a conserved molecular structure for all of the mutants. The proximal residue coordinates, together with the dipolar shifts for proximal side residues, quantitatively yield the orientations of the magnetic susceptibility tensors, whose major axis corresponds to the orientation of the ligand. It is observed that upon reduction of the steric bulk in the V[E11]A mutant, the tilt of the ligand is significantly reduced (∼8°) from that in the wild type (WT) (∼16°), with little change in the direction of tilt. In the case of increased steric bulk at position 68 in the V[E11]I mutant, it is observed that the extent and direction of the tilt are essentially the same as in WT, and it is shown that this is due to the fact that Ile68 is oriented in the pocket with its C5H3 directed away from the iron. The removal of the bulky imidazole side chain in the H[E7]G and H[E7]V mutants leaves the extent of tilt unchanged from that in WT, but with a direction of tilt rotated by ∼40° that has been interpreted in terms of the energy surface of the heme pocket [Rajarathnam, K., et al. (1993) Biochemistry 32, 5670-5680]. Hence, the E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand, respectively. In the double mutants, the influences of the E7 and E11 substitutions are essentially additive, with the ligand tilt adopting the direction of the H[E7]G single mutant and the extent of tilt of the V[E11]A or V[E11]I single mutant. The extent of the ligand tilt determined herein for the various cyanometmyoglobin mutants does not correlate with the kinetics or thermodynamics of CO ligation, indicating that factors other than steric effects, such as polarity of the heme pocket, play an important role in modulating ligand binding.

AB - The amino acids in the heme pocket of sperm whale myoglobin single E11 and double E7 and E11 point mutants in the metcyano form have been assigned by NMR methods to assess the role of steric bulk in modulating ligand tilt. The five mutants investigated are the single mutants His64(E7)→Gly (H[E7]G), Val68(E11)→Ile (V[E11]I), and Val68(E11)→Ala (V[E11]A) and the double mutants His64-(E7)→Gly:Val68(E11)→Ile (H,V[E7,E11]G,I) and His64(E7)→Gly:Val68(E11)→Ala (H,V[E7,E11]G,A). The dipolar (NOESY) contacts on the proximal side of the heme confirm a conserved molecular structure for all of the mutants. The proximal residue coordinates, together with the dipolar shifts for proximal side residues, quantitatively yield the orientations of the magnetic susceptibility tensors, whose major axis corresponds to the orientation of the ligand. It is observed that upon reduction of the steric bulk in the V[E11]A mutant, the tilt of the ligand is significantly reduced (∼8°) from that in the wild type (WT) (∼16°), with little change in the direction of tilt. In the case of increased steric bulk at position 68 in the V[E11]I mutant, it is observed that the extent and direction of the tilt are essentially the same as in WT, and it is shown that this is due to the fact that Ile68 is oriented in the pocket with its C5H3 directed away from the iron. The removal of the bulky imidazole side chain in the H[E7]G and H[E7]V mutants leaves the extent of tilt unchanged from that in WT, but with a direction of tilt rotated by ∼40° that has been interpreted in terms of the energy surface of the heme pocket [Rajarathnam, K., et al. (1993) Biochemistry 32, 5670-5680]. Hence, the E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand, respectively. In the double mutants, the influences of the E7 and E11 substitutions are essentially additive, with the ligand tilt adopting the direction of the H[E7]G single mutant and the extent of tilt of the V[E11]A or V[E11]I single mutant. The extent of the ligand tilt determined herein for the various cyanometmyoglobin mutants does not correlate with the kinetics or thermodynamics of CO ligation, indicating that factors other than steric effects, such as polarity of the heme pocket, play an important role in modulating ligand binding.

UR - http://www.scopus.com/inward/record.url?scp=0028303641&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028303641&partnerID=8YFLogxK

M3 - Article

VL - 33

SP - 5493

EP - 5501

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 18

ER -