ENDOR spectroscopic evidence for the geometry of binding of retro-inverso-N(ω)-nitroarginine-containing dipeptide amides to neuronal nitric oxide synthase

David L. Tierney, Hui Huang, Pavel Martásek, Linda J. Roman, Richard B. Silverman, Brian M. Hoffman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

We describe in detail the use of 35 GHz Mims pulsed 15N and (1,2)H electron - nuclear double resonance (ENDOR) spectroscopy to study the binding of substrates and inhibitors to nitric oxide synthase (NOS). We show that reliable distance estimates, and limited orientation information, can be derived from a small set of data taken near the peak of the absorption mode EPR signal, while more precise orientations require a more extensive data set. The ENDOR approach is then applied to the binding of isoform-selective and non-selective nitroarginine inhibitors. Recently, we reported a family of N(ω)-nitroarginine-containing dipeptide amides as highly selective inhibitors of nNOS (Huang, H. et al. J. Med. Chem. 1999, 42, 3147-3153). Two of the most potent analogues were the retro-inverso-dipeptide amides L-Arg(NO2)-L-Lys-NH2 (LL) and D-Lys-D-Arg(NO2)-NH2 (DD). To rationalize the common selectivities of LL and DD, it was proposed that in both cases the nitroarginine group binds at the heme binding site, therefore requiring one of these molecules to undergo a 180°flip to accommodate such an interaction. The present studies confirm that the dipeptides indeed bind to holo-nNOS quite similarly from the point of view of the nitroguanidine functionality, supporting the earlier interpretation. The data further suggest that a substantial fraction of the DD epimer is distributed among other binding geometries.

Original languageEnglish (US)
Pages (from-to)7869-7875
Number of pages7
JournalJournal of the American Chemical Society
Volume122
Issue number33
DOIs
StatePublished - Aug 23 2000

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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