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

David L. Tierney, Hui Huang, Pavel Martasek, Bettie Sue Siler Masters, Richard B. Silverman, Brian M. Hoffman

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49 Scopus citations

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, N(G)- hydroxy-L-arginine (NOHA), singly labeled with 15N at the hydroxylated nitrogen (denoted N(R)), show that N(R) is held 3.8 Å from the Fe, closer than the corresponding guanidino N of L-Arg (4.05 Å). 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 Å from Fe and very near the heine 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.

Original languageEnglish (US)
Pages (from-to)3704-3710
Number of pages7
JournalBiochemistry
Volume38
Issue number12
DOIs
StatePublished - Mar 23 1999

ASJC Scopus subject areas

  • Biochemistry

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