Substrate-ligand interactions in geobacillus stearothermophilus nitric oxide synthase

Mariam Kabir, Jawahar Sudhamsu, Brian R. Crane, Syun-Ru Yeh, Denis L. Rousseau

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Nitric oxide synthase (NOS) generates NO via a sequential two-step reaction [L-arginine (L-Arg) → N-hydroxy-L-arginine (NOHA) → L-citrulline + NO]. Each step of the reaction follows a distinct mechanism defined by the chemical environment introduced by each substrate bound to the heme active site. The dioxygen complex of the NOS enzyme from a thermophilic bacterium, Geobacillus stearothermophilus (gsNOS), is unusually stable; hence, it provides a unique model for the studies of the mechanistic differences between the two steps of the NOS reaction. By using CO as a structural probe, we found that gsNOS exhibits two conformations in the absence of substrate, as indicated by the presence of two sets of νFe-COC-O modes in the resonance Raman spectra. In the νFe-CO versus νC-O inverse correlation plot, one set of data falls on the correlation line characterized by mammalian NOSs (mNOS), whereas the other set of data lies on a new correlation line defined by a bacterial NOS from Bacillus subtilis (bsNOS), reflecting a difference in the proximal Fe-Cys bond strength in the two conformers of gsNOS. The addition of L-Arg stabilizes the conformer associated with the mNOS correlation line, whereas NOHA stabilizes the conformer associated with the bsNOS correlation line, although both substrates introduce a positive electrostatic potential into the distal heme pocket. To assess how substrate binding affects Fe-Cys bond strength, the frequency of the Fe-Cys stretching mode of gsNOS was monitored by resonance Raman spectroscopy with 363.8 nm excitation. In the substrate-free form, the Fe-Cys stretching mode was detected at 342.5 cm-1, similar to that of bsNOS. The binding of L-Arg and NOHA brings about a small decrease and increase in the Fe-Cys stretching frequency, respectively. The implication of these unique structural features with respect to the oxygen chemistry of NOS is discussed.

Original languageEnglish (US)
Pages (from-to)12389-12397
Number of pages9
JournalBiochemistry
Volume47
Issue number47
DOIs
StatePublished - Nov 25 2008

Fingerprint

Geobacillus stearothermophilus
Nitric Oxide Synthase
Ligands
Carbon Monoxide
Stretching
Substrates
Heme
Arginine
Oxygen
Citrulline
Raman Spectrum Analysis
Bacilli
Bacillus subtilis
Static Electricity
Raman spectroscopy
Conformations
Raman scattering
Electrostatics
Catalytic Domain
Bacteria

ASJC Scopus subject areas

  • Biochemistry

Cite this

Substrate-ligand interactions in geobacillus stearothermophilus nitric oxide synthase. / Kabir, Mariam; Sudhamsu, Jawahar; Crane, Brian R.; Yeh, Syun-Ru; Rousseau, Denis L.

In: Biochemistry, Vol. 47, No. 47, 25.11.2008, p. 12389-12397.

Research output: Contribution to journalArticle

Kabir, Mariam ; Sudhamsu, Jawahar ; Crane, Brian R. ; Yeh, Syun-Ru ; Rousseau, Denis L. / Substrate-ligand interactions in geobacillus stearothermophilus nitric oxide synthase. In: Biochemistry. 2008 ; Vol. 47, No. 47. pp. 12389-12397.
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abstract = "Nitric oxide synthase (NOS) generates NO via a sequential two-step reaction [L-arginine (L-Arg) → N-hydroxy-L-arginine (NOHA) → L-citrulline + NO]. Each step of the reaction follows a distinct mechanism defined by the chemical environment introduced by each substrate bound to the heme active site. The dioxygen complex of the NOS enzyme from a thermophilic bacterium, Geobacillus stearothermophilus (gsNOS), is unusually stable; hence, it provides a unique model for the studies of the mechanistic differences between the two steps of the NOS reaction. By using CO as a structural probe, we found that gsNOS exhibits two conformations in the absence of substrate, as indicated by the presence of two sets of νFe-CO/νC-O modes in the resonance Raman spectra. In the νFe-CO versus νC-O inverse correlation plot, one set of data falls on the correlation line characterized by mammalian NOSs (mNOS), whereas the other set of data lies on a new correlation line defined by a bacterial NOS from Bacillus subtilis (bsNOS), reflecting a difference in the proximal Fe-Cys bond strength in the two conformers of gsNOS. The addition of L-Arg stabilizes the conformer associated with the mNOS correlation line, whereas NOHA stabilizes the conformer associated with the bsNOS correlation line, although both substrates introduce a positive electrostatic potential into the distal heme pocket. To assess how substrate binding affects Fe-Cys bond strength, the frequency of the Fe-Cys stretching mode of gsNOS was monitored by resonance Raman spectroscopy with 363.8 nm excitation. In the substrate-free form, the Fe-Cys stretching mode was detected at 342.5 cm-1, similar to that of bsNOS. The binding of L-Arg and NOHA brings about a small decrease and increase in the Fe-Cys stretching frequency, respectively. The implication of these unique structural features with respect to the oxygen chemistry of NOS is discussed.",
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