ENDOR spectroscopic evidence for the position and structure of NG-hydroxy-L-arginine bound to holo-neuronal nitric oxide synthase

Recently, we used 35 GHz pulsed N-15 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-hound reaction intermediate, N-G-hydroxy-L-arginine (NOHA), singly labeled with N-15 at the hydroxylated nitrogen (denoted N-R), show that N-R is held 3.8 Angstrom from the Fe, closer than the corresponding guanidino N of L-Arg (4.05 Angstrom). H-1,H-2 ENDOR of NOHA bound to holo-nNOS in H2O and D2O discloses the presence of a single resolved exchangeable proton (H1) 4.8 Angstrom 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 N-R-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 N-R and thus of the tautomeric form that is present in the enzyme-NOHA complex. The mechanistic implications of this result are further discussed.