ENDOR spectroscopic evidence for the position and structure of NG-hydroxy-L-arginine bound to holo-neuronal nitric oxide synthase.
Academic Article
Overview
Research
Identity
Additional Document Info
View All
Overview
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.
