title: ENDOR spectroscopic evidence for the position and structure of NG-hydroxy-L-arginine bound to holo-neuronal nitric oxide synthase.
abstract: Recently, we used 35 GHz pulsed 15N ENDOR spectroscopy to determine the position of the reactive guanidino nitrogen of substrate L-arginine relative to the high-spin ferriheme iron of holo-neuronal nitric oxide synthase (nNOS) [Tierney, D. L., et al. (1998) J. Am. Chem. Soc. 120, 2983-2984]. Analogous studies of the enzyme-bound reaction intermediate, NG-hydroxy-L-arginine (NOHA), singly labeled with 15N at the hydroxylated nitrogen (denoted NR), show that NR is held 3.8 A from the Fe, closer than the corresponding guanidino N of L-Arg (4.05 A). 1,2H ENDOR of NOHA bound to holo-nNOS in H2O and D2O discloses the presence of a single resolved exchangeable proton (H1) 4.8 A from Fe and very near the heme normal. The ENDOR data indicate that NOHA does not bind as the resonance-stabilized cation in which the terminal nitrogens share a positive charge. ENDOR-determined structural constraints permit two alternate structural models for the interaction of NOHA with the high-spin heme iron. In one model, H1 is assigned to the O-H proton; in the other, it is the NR-H proton. However, the alternatives differ in the placement of the N-O bond relative to the heme iron. Thus, a combination of the ENDOR data with appropriate diffraction studies can achieve a definitive determination of the protonation state of NR and thus of the tautomeric form that is present in the enzyme-NOHA complex. The mechanistic implications of this result are further discussed.
authors: Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3113, USA.
authors: D L
authors: DL
authors: Tierney
authors: H
authors: H
authors: Huang
authors: P
authors: P
authors: Martasek
authors: B S
authors: BS
authors: Masters
authors: R B
authors: RB
authors: Silverman
authors: B M
authors: BM
authors: Hoffman
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D020035:Holoenzymes / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D011487:Protein Conformation
chemical_list: D020035:Holoenzymes; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I