Probing the Hydrogen Bonding of the Ferrous鈥揘O Heme Center of nNOS by Pulsed Electron Paramagnetic Resonance

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• 作者：Andrei V. Astashkin ; Li Chen ; Bradley O. Elmore ; Deepak Kunwar ; Yubin Miao ; Huiying Li ; Thomas L. Poulos ; Linda J. Roman ; Changjian Feng
• 刊名：Journal of Physical Chemistry A
• 出版年：2015
• 出版时间：June 25, 2015
• 年：2015
• 卷：119
• 期：25
• 页码：6641-6649
• 全文大小：377K
• ISSN：1520-5215

文摘

Oxidation of l-arginine (l-Arg) to nitric oxide (NO) by NO synthase (NOS) takes place at the heme active site. It is of current interest to study structures of the heme species that activates O₂ and transforms the substrate. The NOS ferrous鈥揘O complex is a close mimic of the obligatory ferric (hydro)peroxo intermediate in NOS catalysis. In this work, pulsed electron鈥搉uclear double resonance (ENDOR) spectroscopy was used to probe the hydrogen bonding of the NO ligand in the ferrous鈥揘O heme center of neuronal NOS (nNOS) without a substrate and with l-Arg or N-hydroxy-l-arginine (NOHA) substrates. Unexpectedly, no H-bonding interaction connecting the NO ligand to the active site water molecule or the Arg substrate was detected, in contrast to the results obtained by X-ray crystallography for the Arg-bound nNOS heme domain [Li et al. J. Biol. Inorg. Chem. 2006, 11, 753鈭?68]. The nearby exchangeable proton in both the no-substrate and Arg-containing nNOS samples is located outside the H-bonding range and, on the basis of the obtained structural constraints, can belong to the active site water (or OH). On the contrary, in the NOHA-bound sample, the nearby exchangeable hydrogen forms an H-bond with the NO ligand (on the basis of its distance from the NO ligand and a nonzero isotropic hfi constant), but it does not belong to the active site water molecule because the water oxygen atom (detected by ¹⁷O ENDOR) is too far. This hydrogen should therefore come from the NOHA substrate, which is in agreement with the X-ray crystallography work [Li et al. Biochemistry 2009, 48, 10246鈭?0254]. The nearby nonexchangeable hydrogen atom assigned as H_蔚 of Phe584 was detected in all three samples. This hydrogen atom may have a stabilizing effect on the NO ligand and probably determines its position.