Functional implications of the proximal hydrogen-bonding network in myoglobin: A resonance raman and kinetic study of Leu89, Ser92, His97, and F- helix swap mutants

Eric S. Peterson, Joel M. Friedman, Ellen Y T Chien, Stephen G. Sligar

Research output: Contribution to journalArticle

87 Citations (Scopus)

Abstract

Resonance Raman spectra have been obtained for both the equilibrium deoxy derivative and the 10 ns photoproduct of the CO derivative of several mutants of sperm whale myoglobin. The particular mutations on the F-helix were chosen to expose the role of the proximal hydrogen-bonding network in maintaining the position of the heme, the proximal histidine, and the heme- 7-propionate. In each mutant, one or more hydrogen bonds are altered or eliminated. A careful comparison of the spectra from the equilibrium and transient five coordinate species indicates that the tertiary relaxation after photodissociation is nearly complete within 10 ns, as is the case in the WT protein. The iron-proximal histidine stretching mode (υ(Fe-His)) and several low-frequency propionate-sensitive modes in the Raman spectra reveal the impact of specific disruptions in the hydrogen-bonding network on the heme pocket geometry. Two categories of perturbation are observed with respect to υ(Fe-His): (1) a shift in the peak frequency without a change in line shape and (2) changes in the overall line shape which may or may not be accompanied by a frequency shift. The alterations in the υ(Fe-His) band are interpreted as arising from conformational heterogeneity and local geometrical changes within the pocket, including movement of the heme group, and are discussed in terms of changes in the population distribution as revealed via a curve-fitting analysis. None of the frequency shifts in the υ(Fe-His) band are as large as that reported for the His93Gly(imidazole) mutant, suggesting that the covalent linkage between the heme and His93 plays a crucial role in maintaining the geometry of the proximal pocket. Molecular modeling indicates that the υ(Fe-His) frequency shifts observed in the present study originate from changes in the His93 imidazole ring azimuthal angle. The systematic variations in the interactions of the heme-7-propionate in the mutants have exposed several properties of the propionate-sensitive Raman bands. The frequencies of υ9 (the 240 cm-1 shoulder on the υ(Fe- His) band) and δ(c(β)c(c)c(d)) at ~370 cm-1 appear to be correlated. A decrease in hydrogen-bond strength to this propionate in response to changes in stereochemistry or degree of disorder is associated with a decrease in the frequency of both υ9 and δ(c(β)c(c)c(d)). The mutations that cause a weakening of the hydrogen bonding to the heme-7-propionate also result in changes in υ(Fe-His) which are interpreted as evidence that this propionate participates in the anchoring of the heme within the heme pocket. Changes in γ7 at ~300 cm-1, γ6 at ~335 cm-1, and υ8 at ~342 cm-1 are discussed in terms of pocket disorder. A titration from pH 5.1 to 7.4 suggests that His97 is protonated in the WT protein by pH 5.1. Geminate- rebinding studies on these mutants indicate that disruption of the hydrogen- bonding network has only modest effects on ligand-binding kinetics, suggesting that the role of the hydrogen-bonding network may be one of maintaining heme pocket stability rather than of specific protein function.

Original languageEnglish (US)
Pages (from-to)12301-12319
Number of pages19
JournalBiochemistry
Volume37
Issue number35
DOIs
StatePublished - Sep 1 1998

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Myoglobin
Hydrogen Bonding
Heme
Hydrogen bonds
Propionates
Kinetics
Histidine
Raman scattering
Hydrogen
Sperm Whale
Population distribution
Derivatives
Photodissociation
Mutation
Stereochemistry
Proteins
Molecular modeling
Geometry
Curve fitting
Carbon Monoxide

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional implications of the proximal hydrogen-bonding network in myoglobin : A resonance raman and kinetic study of Leu89, Ser92, His97, and F- helix swap mutants. / Peterson, Eric S.; Friedman, Joel M.; Chien, Ellen Y T; Sligar, Stephen G.

In: Biochemistry, Vol. 37, No. 35, 01.09.1998, p. 12301-12319.

Research output: Contribution to journalArticle

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abstract = "Resonance Raman spectra have been obtained for both the equilibrium deoxy derivative and the 10 ns photoproduct of the CO derivative of several mutants of sperm whale myoglobin. The particular mutations on the F-helix were chosen to expose the role of the proximal hydrogen-bonding network in maintaining the position of the heme, the proximal histidine, and the heme- 7-propionate. In each mutant, one or more hydrogen bonds are altered or eliminated. A careful comparison of the spectra from the equilibrium and transient five coordinate species indicates that the tertiary relaxation after photodissociation is nearly complete within 10 ns, as is the case in the WT protein. The iron-proximal histidine stretching mode (υ(Fe-His)) and several low-frequency propionate-sensitive modes in the Raman spectra reveal the impact of specific disruptions in the hydrogen-bonding network on the heme pocket geometry. Two categories of perturbation are observed with respect to υ(Fe-His): (1) a shift in the peak frequency without a change in line shape and (2) changes in the overall line shape which may or may not be accompanied by a frequency shift. The alterations in the υ(Fe-His) band are interpreted as arising from conformational heterogeneity and local geometrical changes within the pocket, including movement of the heme group, and are discussed in terms of changes in the population distribution as revealed via a curve-fitting analysis. None of the frequency shifts in the υ(Fe-His) band are as large as that reported for the His93Gly(imidazole) mutant, suggesting that the covalent linkage between the heme and His93 plays a crucial role in maintaining the geometry of the proximal pocket. Molecular modeling indicates that the υ(Fe-His) frequency shifts observed in the present study originate from changes in the His93 imidazole ring azimuthal angle. The systematic variations in the interactions of the heme-7-propionate in the mutants have exposed several properties of the propionate-sensitive Raman bands. The frequencies of υ9 (the 240 cm-1 shoulder on the υ(Fe- His) band) and δ(c(β)c(c)c(d)) at ~370 cm-1 appear to be correlated. A decrease in hydrogen-bond strength to this propionate in response to changes in stereochemistry or degree of disorder is associated with a decrease in the frequency of both υ9 and δ(c(β)c(c)c(d)). The mutations that cause a weakening of the hydrogen bonding to the heme-7-propionate also result in changes in υ(Fe-His) which are interpreted as evidence that this propionate participates in the anchoring of the heme within the heme pocket. Changes in γ7 at ~300 cm-1, γ6 at ~335 cm-1, and υ8 at ~342 cm-1 are discussed in terms of pocket disorder. A titration from pH 5.1 to 7.4 suggests that His97 is protonated in the WT protein by pH 5.1. Geminate- rebinding studies on these mutants indicate that disruption of the hydrogen- bonding network has only modest effects on ligand-binding kinetics, suggesting that the role of the hydrogen-bonding network may be one of maintaining heme pocket stability rather than of specific protein function.",
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T1 - Functional implications of the proximal hydrogen-bonding network in myoglobin

T2 - A resonance raman and kinetic study of Leu89, Ser92, His97, and F- helix swap mutants

AU - Peterson, Eric S.

AU - Friedman, Joel M.

AU - Chien, Ellen Y T

AU - Sligar, Stephen G.

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N2 - Resonance Raman spectra have been obtained for both the equilibrium deoxy derivative and the 10 ns photoproduct of the CO derivative of several mutants of sperm whale myoglobin. The particular mutations on the F-helix were chosen to expose the role of the proximal hydrogen-bonding network in maintaining the position of the heme, the proximal histidine, and the heme- 7-propionate. In each mutant, one or more hydrogen bonds are altered or eliminated. A careful comparison of the spectra from the equilibrium and transient five coordinate species indicates that the tertiary relaxation after photodissociation is nearly complete within 10 ns, as is the case in the WT protein. The iron-proximal histidine stretching mode (υ(Fe-His)) and several low-frequency propionate-sensitive modes in the Raman spectra reveal the impact of specific disruptions in the hydrogen-bonding network on the heme pocket geometry. Two categories of perturbation are observed with respect to υ(Fe-His): (1) a shift in the peak frequency without a change in line shape and (2) changes in the overall line shape which may or may not be accompanied by a frequency shift. The alterations in the υ(Fe-His) band are interpreted as arising from conformational heterogeneity and local geometrical changes within the pocket, including movement of the heme group, and are discussed in terms of changes in the population distribution as revealed via a curve-fitting analysis. None of the frequency shifts in the υ(Fe-His) band are as large as that reported for the His93Gly(imidazole) mutant, suggesting that the covalent linkage between the heme and His93 plays a crucial role in maintaining the geometry of the proximal pocket. Molecular modeling indicates that the υ(Fe-His) frequency shifts observed in the present study originate from changes in the His93 imidazole ring azimuthal angle. The systematic variations in the interactions of the heme-7-propionate in the mutants have exposed several properties of the propionate-sensitive Raman bands. The frequencies of υ9 (the 240 cm-1 shoulder on the υ(Fe- His) band) and δ(c(β)c(c)c(d)) at ~370 cm-1 appear to be correlated. A decrease in hydrogen-bond strength to this propionate in response to changes in stereochemistry or degree of disorder is associated with a decrease in the frequency of both υ9 and δ(c(β)c(c)c(d)). The mutations that cause a weakening of the hydrogen bonding to the heme-7-propionate also result in changes in υ(Fe-His) which are interpreted as evidence that this propionate participates in the anchoring of the heme within the heme pocket. Changes in γ7 at ~300 cm-1, γ6 at ~335 cm-1, and υ8 at ~342 cm-1 are discussed in terms of pocket disorder. A titration from pH 5.1 to 7.4 suggests that His97 is protonated in the WT protein by pH 5.1. Geminate- rebinding studies on these mutants indicate that disruption of the hydrogen- bonding network has only modest effects on ligand-binding kinetics, suggesting that the role of the hydrogen-bonding network may be one of maintaining heme pocket stability rather than of specific protein function.

AB - Resonance Raman spectra have been obtained for both the equilibrium deoxy derivative and the 10 ns photoproduct of the CO derivative of several mutants of sperm whale myoglobin. The particular mutations on the F-helix were chosen to expose the role of the proximal hydrogen-bonding network in maintaining the position of the heme, the proximal histidine, and the heme- 7-propionate. In each mutant, one or more hydrogen bonds are altered or eliminated. A careful comparison of the spectra from the equilibrium and transient five coordinate species indicates that the tertiary relaxation after photodissociation is nearly complete within 10 ns, as is the case in the WT protein. The iron-proximal histidine stretching mode (υ(Fe-His)) and several low-frequency propionate-sensitive modes in the Raman spectra reveal the impact of specific disruptions in the hydrogen-bonding network on the heme pocket geometry. Two categories of perturbation are observed with respect to υ(Fe-His): (1) a shift in the peak frequency without a change in line shape and (2) changes in the overall line shape which may or may not be accompanied by a frequency shift. The alterations in the υ(Fe-His) band are interpreted as arising from conformational heterogeneity and local geometrical changes within the pocket, including movement of the heme group, and are discussed in terms of changes in the population distribution as revealed via a curve-fitting analysis. None of the frequency shifts in the υ(Fe-His) band are as large as that reported for the His93Gly(imidazole) mutant, suggesting that the covalent linkage between the heme and His93 plays a crucial role in maintaining the geometry of the proximal pocket. Molecular modeling indicates that the υ(Fe-His) frequency shifts observed in the present study originate from changes in the His93 imidazole ring azimuthal angle. The systematic variations in the interactions of the heme-7-propionate in the mutants have exposed several properties of the propionate-sensitive Raman bands. The frequencies of υ9 (the 240 cm-1 shoulder on the υ(Fe- His) band) and δ(c(β)c(c)c(d)) at ~370 cm-1 appear to be correlated. A decrease in hydrogen-bond strength to this propionate in response to changes in stereochemistry or degree of disorder is associated with a decrease in the frequency of both υ9 and δ(c(β)c(c)c(d)). The mutations that cause a weakening of the hydrogen bonding to the heme-7-propionate also result in changes in υ(Fe-His) which are interpreted as evidence that this propionate participates in the anchoring of the heme within the heme pocket. Changes in γ7 at ~300 cm-1, γ6 at ~335 cm-1, and υ8 at ~342 cm-1 are discussed in terms of pocket disorder. A titration from pH 5.1 to 7.4 suggests that His97 is protonated in the WT protein by pH 5.1. Geminate- rebinding studies on these mutants indicate that disruption of the hydrogen- bonding network has only modest effects on ligand-binding kinetics, suggesting that the role of the hydrogen-bonding network may be one of maintaining heme pocket stability rather than of specific protein function.

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