Substrate- and Isoform-Specific Dioxygen Complexes of Nitric Oxide Synthase
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- 作者：David Li ; Mariam Kabir ; Dennis J. Stuehr ; Denis L. Rousseau ; Syun-Ru Yeh
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：May 30, 2007
- 年：2007
- 卷：129
- 期：21
- 页码：6943 - 6951
- 全文大小：282K
- 年卷期：v.129,no.21(May 30, 2007)
- ISSN：1520-5126
文摘
Nitric oxide synthase (NOS) catalyzes the formation of NO via a consecutive two-step reaction.In the first step, L-arginine (Arg) is converted to N-hydroxy-L-arginine (NOHA). In the second step, NOHAis further converted to citrulline and nitric oxide (NO). To assess the mechanistic differences between thetwo steps of the reaction, we have used resonance Raman spectroscopy combined with a homemadecontinuous-flow rapid solution mixer to study the structural properties of the metastable dioxygen-boundcomplexes of the oxygenase domain of inducible NOS (iNOSoxy). We identified the O-O stretching frequencyof the substrate-free enzyme at 1133 cm-1. This frequency is insensitive to the presence of tetrahydrobiopterin, but it shifts to 1126 cm-1 upon binding of Arg, which we attribute to H-bonding interactions to theterminal oxygen atom of the heme iron-bound dioxygen. In contrast, the addition of NOHA to the enzymedid not bring about a shift in the frequency of the O-O stretching mode, because, unlike Arg, there is noH-bond associated with the terminal oxygen atom of the dioxygen. The substrate-specific H-bondinginteractions play a critical role in determining the fate of the key peroxy intermediate. In the first step of thereaction, the H-bonds facilitate the rupture of the O-O bond, leading to the formation of the active ferrylspecies, which is essential for the oxidation of the Arg. On the other hand, in the second step of the reaction,the absence of the H-bonds prevents the premature O-O bond cleavage, such that the peroxy intermediatecan perform a nucleophilic addition reaction to the substrate, NOHA.