jp5b01804_si_001.pdf (1.26 MB)
Probing the Hydrogen Bonding of the Ferrous–NO Heme Center of nNOS by Pulsed Electron Paramagnetic Resonance
journal contribution
posted on 2015-06-25, 00:00 authored by Andrei
V. Astashkin, Li Chen, Bradley O. Elmore, Deepak Kunwar, Yubin Miao, Huiying Li, Thomas L. Poulos, Linda J. Roman, Changjian FengOxidation of l-arginine
(l-Arg) to nitric oxide
(NO) by NO synthase (NOS) takes place at the heme active site. It
is of current interest to study structures of the heme species that
activates O2 and transforms the substrate. The NOS ferrous–NO
complex is a close mimic of the obligatory ferric (hydro)peroxo intermediate
in NOS catalysis. In this work, pulsed electron–nuclear double
resonance (ENDOR) spectroscopy was used to probe the hydrogen bonding
of the NO ligand in the ferrous–NO heme center of neuronal
NOS (nNOS) without a substrate and with l-Arg or N-hydroxy-l-arginine (NOHA) substrates. Unexpectedly,
no H-bonding interaction connecting the NO ligand to the active site
water molecule or the Arg substrate was detected, in contrast to the
results obtained by X-ray crystallography for the Arg-bound nNOS heme
domain [Li et al. J. Biol. Inorg.
Chem. 2006, 11, 753−768]. The nearby exchangeable proton
in both the no-substrate and Arg-containing nNOS samples is located
outside the H-bonding range and, on the basis of the obtained structural
constraints, can belong to the active site water (or OH). On the contrary, in the NOHA-bound sample, the nearby exchangeable
hydrogen forms an H-bond with the NO ligand (on the basis of its distance
from the NO ligand and a nonzero isotropic hfi constant),
but it does not belong to the active site water molecule because the
water oxygen atom (detected by 17O ENDOR) is too far. This
hydrogen should therefore come from the NOHA substrate, which is in
agreement with the X-ray crystallography work [Li et al. Biochemistry 2009, 48, 10246−10254]. The nearby nonexchangeable hydrogen atom assigned as
Hε of Phe584 was detected in all three samples. This
hydrogen atom may have a stabilizing effect on the NO ligand and probably
determines its position.
History
Usage metrics
Categories
Keywords
17 O ENDORheme specieswater oxygen atomligandNOHA substrateH εnNOSsite water moleculehydrogen formscrystallographyPulsed Electron Paramagnetic ResonanceOxidationactivates O 2Arg substrateferroubasisPhe 584hydrogen atomnonexchangeable hydrogen atomLisamplesite waterNOS catalysisstudy structuresHydrogen Bondingnitric oxide
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC