Probing the Hydrogen Bonding of the Ferrous-NO Heme Center of nNOS by Pulsed Electron Paramagnetic Resonance

Oxidation 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 H 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.