Substrate−Ligand Interactions in Geobacillus stearothermophilus Nitric Oxide Synthase

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• 作者：Mariam Kabir ; Jawahar Sudhamsu ; Brian R. Crane ; Syun-Ru Yeh ; Denis L. Rousseau
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：November 25, 2008
• 年：2008
• 卷：47
• 期：47
• 页码：12389-12397
• 全文大小：347K
• 年卷期：v.47,no.47(November 25, 2008)
• ISSN：1520-4995

文摘

Nitric oxide synthase (NOS) generates NO via a sequential two-step reaction [lass="smallcaps">l-arginine (lass="smallcaps">l-Arg) → N-hydroxy-lass="smallcaps">l-arginine (NOHA) → lass="smallcaps">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 ν_Fe−CO/ν_C−O modes in the resonance Raman spectra. In the ν_Fe−CO versus ν_C−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 lass="smallcaps">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 lass="smallcaps">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.