Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide, and fluoride to the rhizobial heme-based oxygen sensor FixL

Wade C. Winkler, Gonzalo Gonzalez, Jonathan B. Wittenberg, Russ Hille, Naveen Dakappagari, Anand Jacob, Leyla A. Gonzalez, Marie A. Gilles-Gonzalez

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

Background: The FixL protein is a heme-based sensor. Binding of oxygen to a unique heme domain inhibits a kinase domain of the type found in two- component regulators. Oxygen association is slow, but the dissociation rate is comparable to that of myoglobins. We have probed the size and chemistry of the FixL heme pocket by measuring the affinities, on rates and off rates for a wide variety of ferric heme ligands. Cyanide, but not fluoride, regulates the kinase activity. To examine how the sensory heme domain interacts with the kinase, we asked how the presence of the kinase domain affects ligand binding. Results: The affinities of ferric FixL for heme ligands follow the same trend as their pK(a) values: cyanide > 4-methyl imidazole > imidazole > fluoride > azide >> thiocyanate. The association rates follow the reverse trend. Striking differences from myoglobin include a 6-fold greater affinity for, and faster binding to, the bulky ligand imidazole, a 14-fold faster on rate for nitric oxide, a 2 800-fold lower affinity for azide, and a complete failure to bind thiocyanate. The presence of the kinase domain does not alter the affinity or binding kinetics of the high-spin ligand fluoride, but affects the off rates of other ligands. The EPR spectrum shows a characteristic pentacoordinate nitrosyl heme, indicating that the Fe-His bond in FixL is strained. Conclusions: The importance of ligand deprotonation to the on rates and the fact that large ligands bind readily indicate that the heme pocket is open and apolar. Ligand basicity strongly influences the strength of binding. The destabilization of inhibitory ligands by the presence of the kinase domain is consistent with a 'load' imposed by coupling to the inactivating mechanism.

Original languageEnglish (US)
Pages (from-to)841-850
Number of pages10
JournalChemistry and Biology
Volume3
Issue number10
StatePublished - Oct 1996

Fingerprint

Oxygen sensors
Azides
Cyanides
Heme
Fluorides
Nitric Oxide
Oxygen
Ligands
Phosphotransferases
Myoglobin
imidazole
Association reactions
Deprotonation
Alkalinity
Paramagnetic resonance

Keywords

  • EPR
  • FixL
  • myoglobin
  • nitric oxide
  • oxygen sensor

ASJC Scopus subject areas

  • Organic Chemistry

Cite this

Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide, and fluoride to the rhizobial heme-based oxygen sensor FixL. / Winkler, Wade C.; Gonzalez, Gonzalo; Wittenberg, Jonathan B.; Hille, Russ; Dakappagari, Naveen; Jacob, Anand; Gonzalez, Leyla A.; Gilles-Gonzalez, Marie A.

In: Chemistry and Biology, Vol. 3, No. 10, 10.1996, p. 841-850.

Research output: Contribution to journalArticle

Winkler, WC, Gonzalez, G, Wittenberg, JB, Hille, R, Dakappagari, N, Jacob, A, Gonzalez, LA & Gilles-Gonzalez, MA 1996, 'Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide, and fluoride to the rhizobial heme-based oxygen sensor FixL', Chemistry and Biology, vol. 3, no. 10, pp. 841-850.
Winkler, Wade C. ; Gonzalez, Gonzalo ; Wittenberg, Jonathan B. ; Hille, Russ ; Dakappagari, Naveen ; Jacob, Anand ; Gonzalez, Leyla A. ; Gilles-Gonzalez, Marie A. / Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide, and fluoride to the rhizobial heme-based oxygen sensor FixL. In: Chemistry and Biology. 1996 ; Vol. 3, No. 10. pp. 841-850.
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abstract = "Background: The FixL protein is a heme-based sensor. Binding of oxygen to a unique heme domain inhibits a kinase domain of the type found in two- component regulators. Oxygen association is slow, but the dissociation rate is comparable to that of myoglobins. We have probed the size and chemistry of the FixL heme pocket by measuring the affinities, on rates and off rates for a wide variety of ferric heme ligands. Cyanide, but not fluoride, regulates the kinase activity. To examine how the sensory heme domain interacts with the kinase, we asked how the presence of the kinase domain affects ligand binding. Results: The affinities of ferric FixL for heme ligands follow the same trend as their pK(a) values: cyanide > 4-methyl imidazole > imidazole > fluoride > azide >> thiocyanate. The association rates follow the reverse trend. Striking differences from myoglobin include a 6-fold greater affinity for, and faster binding to, the bulky ligand imidazole, a 14-fold faster on rate for nitric oxide, a 2 800-fold lower affinity for azide, and a complete failure to bind thiocyanate. The presence of the kinase domain does not alter the affinity or binding kinetics of the high-spin ligand fluoride, but affects the off rates of other ligands. The EPR spectrum shows a characteristic pentacoordinate nitrosyl heme, indicating that the Fe-His bond in FixL is strained. Conclusions: The importance of ligand deprotonation to the on rates and the fact that large ligands bind readily indicate that the heme pocket is open and apolar. Ligand basicity strongly influences the strength of binding. The destabilization of inhibitory ligands by the presence of the kinase domain is consistent with a 'load' imposed by coupling to the inactivating mechanism.",
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T1 - Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide, and fluoride to the rhizobial heme-based oxygen sensor FixL

AU - Winkler, Wade C.

AU - Gonzalez, Gonzalo

AU - Wittenberg, Jonathan B.

AU - Hille, Russ

AU - Dakappagari, Naveen

AU - Jacob, Anand

AU - Gonzalez, Leyla A.

AU - Gilles-Gonzalez, Marie A.

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N2 - Background: The FixL protein is a heme-based sensor. Binding of oxygen to a unique heme domain inhibits a kinase domain of the type found in two- component regulators. Oxygen association is slow, but the dissociation rate is comparable to that of myoglobins. We have probed the size and chemistry of the FixL heme pocket by measuring the affinities, on rates and off rates for a wide variety of ferric heme ligands. Cyanide, but not fluoride, regulates the kinase activity. To examine how the sensory heme domain interacts with the kinase, we asked how the presence of the kinase domain affects ligand binding. Results: The affinities of ferric FixL for heme ligands follow the same trend as their pK(a) values: cyanide > 4-methyl imidazole > imidazole > fluoride > azide >> thiocyanate. The association rates follow the reverse trend. Striking differences from myoglobin include a 6-fold greater affinity for, and faster binding to, the bulky ligand imidazole, a 14-fold faster on rate for nitric oxide, a 2 800-fold lower affinity for azide, and a complete failure to bind thiocyanate. The presence of the kinase domain does not alter the affinity or binding kinetics of the high-spin ligand fluoride, but affects the off rates of other ligands. The EPR spectrum shows a characteristic pentacoordinate nitrosyl heme, indicating that the Fe-His bond in FixL is strained. Conclusions: The importance of ligand deprotonation to the on rates and the fact that large ligands bind readily indicate that the heme pocket is open and apolar. Ligand basicity strongly influences the strength of binding. The destabilization of inhibitory ligands by the presence of the kinase domain is consistent with a 'load' imposed by coupling to the inactivating mechanism.

AB - Background: The FixL protein is a heme-based sensor. Binding of oxygen to a unique heme domain inhibits a kinase domain of the type found in two- component regulators. Oxygen association is slow, but the dissociation rate is comparable to that of myoglobins. We have probed the size and chemistry of the FixL heme pocket by measuring the affinities, on rates and off rates for a wide variety of ferric heme ligands. Cyanide, but not fluoride, regulates the kinase activity. To examine how the sensory heme domain interacts with the kinase, we asked how the presence of the kinase domain affects ligand binding. Results: The affinities of ferric FixL for heme ligands follow the same trend as their pK(a) values: cyanide > 4-methyl imidazole > imidazole > fluoride > azide >> thiocyanate. The association rates follow the reverse trend. Striking differences from myoglobin include a 6-fold greater affinity for, and faster binding to, the bulky ligand imidazole, a 14-fold faster on rate for nitric oxide, a 2 800-fold lower affinity for azide, and a complete failure to bind thiocyanate. The presence of the kinase domain does not alter the affinity or binding kinetics of the high-spin ligand fluoride, but affects the off rates of other ligands. The EPR spectrum shows a characteristic pentacoordinate nitrosyl heme, indicating that the Fe-His bond in FixL is strained. Conclusions: The importance of ligand deprotonation to the on rates and the fact that large ligands bind readily indicate that the heme pocket is open and apolar. Ligand basicity strongly influences the strength of binding. The destabilization of inhibitory ligands by the presence of the kinase domain is consistent with a 'load' imposed by coupling to the inactivating mechanism.

KW - EPR

KW - FixL

KW - myoglobin

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KW - oxygen sensor

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