Pulsed ENDOR determination of the arginine location in the ferrous-NO form of neuronal NOS.
Mammalian nitric oxide synthases (NOSs) are enzymes responsible for oxidation of L-arginine (L-Arg) to nitric oxide (NO). Mechanisms of reactions at the catalytic heme site are not well understood, and it is of current interest to study structures of the heme species that activates O(2) 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) was used to probe the position of the l-Arg substrate at the NO(•)-coordinated ferrous heme center(s) in the oxygenase domain of rat neuronal NOS (nNOS). The analysis of (2)H and (15)N ENDOR spectra of samples containing d(7)- or guanidino-(15)N(2) labeled L-Arg has resulted in distance estimates for the nearby guanidino nitrogen and the nearby proton (deuteron) at C(δ). The L-Arg position was found to be noticeably different from that in the X-ray crystal structure of nNOS ferrous-NO complex [Li et al. J. Biol. Inorg. Chem.2006, 11, 753-768], with the nearby guanidino nitrogen being ~0.5 Å closer to, and the nearby H(δ) about 1 Å further from, the NO ligand than in the X-ray structure. The difference might be related to the structural constraints imposed on the protein by the crystal. Importantly, in spite of its closer position, the guanidino nitrogen does not form a hydrogen bond with the NO ligand, as evidenced by the absence of significant isotropic hfi constant for N(g1). This is consistent with the previous reports that it is not the L-Arg substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (NO and O(2)) bound to the heme.
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
Andrei V
AV
Astashkin
Bradley O
BO
Elmore
Li
L
Chen
Weihong
W
Fan
J Guy
JG
Guillemette
Changjian
C
Feng
D000818:Animals; D001120:Arginine / Q000032:analysis* / Q000378:metabolism; D055162:Biocatalysis; D004578:Electron Spin Resonance Spectroscopy; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats
D005296:Ferrous Compounds; D009569:Nitric Oxide; D001120:Arginine; D052248:Nitric Oxide Synthase Type I