Kinetics and equilibria in ligand binding by nitrophorins 1-4: Evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap

J. F. Andersen, X. D. Ding, C. Balfour, T. Kh Shokhireva, D. E. Champagne, F. A. Walker, William "Bill" Montfort

Research output: Contribution to journalArticle

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Abstract

Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 μM-1 s-1) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows (~50 s-1), and the stabilized final complexes are resistant to autoreduction (E°= +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s-1), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s-1). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.

Original languageEnglish (US)
Pages (from-to)10118-10131
Number of pages14
JournalBiochemistry
Volume39
Issue number33
DOIs
StatePublished - Aug 22 2000

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Nitric Oxide
Stabilization
Ligands
Kinetics
Metmyoglobin
Association reactions
Hydrogen
Rate constants
Rhodnius
Burial
nitrophorin
Blood Coagulation
Coagulation
Thermodynamics
Histamine
Insects
Blood Proteins
Hydrogen bonds
Electrodes
Blood

ASJC Scopus subject areas

  • Biochemistry

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Kinetics and equilibria in ligand binding by nitrophorins 1-4 : Evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap. / Andersen, J. F.; Ding, X. D.; Balfour, C.; Shokhireva, T. Kh; Champagne, D. E.; Walker, F. A.; Montfort, William "Bill".

In: Biochemistry, Vol. 39, No. 33, 22.08.2000, p. 10118-10131.

Research output: Contribution to journalArticle

Andersen, J. F. ; Ding, X. D. ; Balfour, C. ; Shokhireva, T. Kh ; Champagne, D. E. ; Walker, F. A. ; Montfort, William "Bill". / Kinetics and equilibria in ligand binding by nitrophorins 1-4 : Evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap. In: Biochemistry. 2000 ; Vol. 39, No. 33. pp. 10118-10131.
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abstract = "Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 μM-1 s-1) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows (~50 s-1), and the stabilized final complexes are resistant to autoreduction (E°= +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s-1), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s-1). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.",
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T2 - Evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap

AU - Andersen, J. F.

AU - Ding, X. D.

AU - Balfour, C.

AU - Shokhireva, T. Kh

AU - Champagne, D. E.

AU - Walker, F. A.

AU - Montfort, William "Bill"

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N2 - Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 μM-1 s-1) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows (~50 s-1), and the stabilized final complexes are resistant to autoreduction (E°= +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s-1), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s-1). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.

AB - Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 μM-1 s-1) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows (~50 s-1), and the stabilized final complexes are resistant to autoreduction (E°= +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s-1), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s-1). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.

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