title: Probing the Hydrogen Bonding of the Ferrous-NO Heme Center of nNOS by Pulsed Electron Paramagnetic Resonance.
abstract: 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-NO complex is a close mimic of the obligatory ferric (hydro)peroxo intermediate in NOS catalysis. In this work, pulsed electron-nuclear double resonance (ENDOR) spectroscopy was used to probe the hydrogen bonding of the NO ligand in the ferrous-NO 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, 753-768]. 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 (17)O 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, 10246-10254]. The nearby nonexchangeable hydrogen atom assigned as H(ε) 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.
authors.affiliation: †Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States.
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Astashkin
authors.affiliation: ‡College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, United States.
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.affiliation: ‡College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, United States.
authors.forename: Bradley O
authors.initials: BO
authors.lastname: Elmore
authors.affiliation: ‡College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, United States.
authors.forename: Deepak
authors.initials: D
authors.lastname: Kunwar
authors.affiliation: ‡College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, United States.
authors.forename: Yubin
authors.initials: Y
authors.lastname: Miao
authors.affiliation: §Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697-3900, United States.
authors.forename: Huiying
authors.initials: H
authors.lastname: Li
authors.affiliation: §Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697-3900, United States.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Poulos
authors.affiliation: ∥Department of Biochemistry, University of Texas Health Science Center in San Antonio, San Antonio, Texas 78229, United States.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Roman
authors.affiliation: ‡College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, United States.
authors.forename: Changjian
authors.initials: C
authors.lastname: Feng
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D009569:Nitric Oxide / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry*; D011522:Protons; D051381:Rats; D014867:Water / Q000737:chemistry
chemical_list: D011522:Protons; D014867:Water; D009569:Nitric Oxide; D006418:Heme; D006859:Hydrogen; D001120:Arginine; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat