ENDOR spectroscopic evidence for the geometry of binding of retro-inverso-N-omega-nitroarginine-containing dipeptide amides to neuronal nitric oxide synthase
Dl. Tierney et al., ENDOR spectroscopic evidence for the geometry of binding of retro-inverso-N-omega-nitroarginine-containing dipeptide amides to neuronal nitric oxide synthase, J AM CHEM S, 122(33), 2000, pp. 7869-7875
We describe in detail the use of 35 GHz Mims pulsed N-15 and H-1,H-2 electr
on-nuclear double resonance (ENDOR) spectroscopy to study the binding of su
bstrates and inhibitors to nitric oxide synthase (NOS). We show that reliab
le distance estimates, and limited orientation information, can be derived
from a small set of data taken near the peak of the absorption mode EPR sig
nal, 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 Nw-n
itroarginine-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-N
H2 (LL) and D-Lys-D-Arg(NO2)-NH2 (DD). To rationalize the common selectivit
ies of LL and DD, it was proposed that in both cases the nitroarginine grou
p binds at the heme binding site, therefore requiring one of these molecule
s to undergo a 180 degrees flip to accommodate such an interaction. The pre
sent studies confirm that the dipeptides indeed bind to holo-nNOS quite sim
ilarly from the point of view of the nitroguanidine functionality, supporti
ng the earlier interpretation. The data further suggest that a substantial
fraction of the DD epimer is distributed among other binding geometries.