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

Abstract

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 Hɛ 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.

Interdomain Interactions Modulate the Active Site Dynamics of Human Inducible Nitric Oxide Synthase

“…When the residue is mutated in human iNOS (E546N), the FMN–heme IET is noticeably diminished due to a larger entropic activation barrier compared to the wild type . Pulsed EPR data indicate that the charge neutralization mutation decreases the frequency of contact between the FMN and heme domains. , In agreement, a charge reversal mutation at another surface residue in murine iNOS (E545 R ) leads to perturbation of the interaction between the FMN and heme domains …”

“…19 Pulsed EPR data indicate that the charge neutralization mutation decreases the frequency of contact between the FMN and heme domains. 19,20 In agreement, a charge reversal mutation at another surface residue in murine iNOS (E545R) leads to perturbation of the interaction between the FMN and heme domains. 13 While evidence increasingly supports that association of the FMN and heme domains is required for establishing a productive pathway for the FMN−heme IET, little is known about whether and how the complexation otherwise impacts the oxygenase domain and its functionality for subsequent chemistry at the heme center.…”