Substrate Binding-Induced Changes in the EPR Spectra of the Ferrous Nitric Oxide Complexes of Neuronal Nitric Oxide Synthase

Abstract

A versatile diatomic physiological messenger, nitric oxide (NO), is biosynthesized by a group of flavo-heme enzymes, the nitric oxide synthases. We have examined the active site of the neuronal isoform by EPR spectroscopy of the ferrous nitric oxide complex. The nitric oxide complex of the substrate-free enzyme exhibits a cytochrome P450-type EPR spectrum typical of a hexacoordinate NO-heme complex with a non-nitrogenous proximal axial heme ligand. The NO complex of the substrate-free enzyme is rather unstable and spontaneously converts to a cytochrome P420 type pentacoordinate denatured form. Binding of L-arginine (l-Arg) enhances the stability of the hexacoordinate NO form. The EPR spectrum of the NO adduct of the enzyme-substrate complex has an increased g-anisotropy and well-resolved hyperfine couplings due to the 14N of nitric oxide. Significant perturbations in the NO EPR spectrum were observed upon Nomega-monomethyl-L-Arg and Nomega-hydroxy-L-Arg binding. The perturbations in the EPR spectrum indicate that L-Arg and its derivatives bind on the distal site of the heme in very close proximity to the bound NO to cause alterations in the heme-NO coordination structure. Interactions between the bound NO and the substrate or its analogues appear to affect the Fe-NO geometry, resulting in the observed spectral changes. We infer that analogous interactions with oxygen might be involved in the hydroxylation events during enzyme catalysis of nitric oxide synthase.

Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

“…At first sight the EPR spectrum suggests a five-coordinate NO complex with a small admixture of six-coordinate Fe(II)⅐NO (34,37,38). As already reported previously (13), no intermediate with a Soret band at 436 nm was observed, confirming the absence of six-coordinate thiolate-ligated ferrous⅐NO heme.…”

“…8B), although the signal intensity was reduced, the shape became distorted, and a minor signal at g ϭ 2.038 appeared. A minor signal at g ϭ 2.03 has been observed before under similar conditions and may be due to a species with different Fe⅐NO geometry (34). We found no trace of the Fe(II)⅐NO complex observed with 4-amino-BH4 and NHA after single-turnover.…”

“…When NO was added to an anaerobic EPR sample of dithionitereduced eNOS oxy (55 M) in the presence of Arg and 4-amino-BH4 (1.5 mM each), a spectrum typical of six-coordinate Fe(II)⅐NO was generated with shape and parameters (g x 2.083, g z 2.005, g y 1.968; A z 20.2 G, A y 11.3 G, Fig. 8A) virtually identical to previously published values (34). Under aerobic conditions the same species dominated the EPR spectrum (Fig.…”

“…It has been demonstrated that Arg, NHA, and BH4 stabilize the six-coordinate form of the ferrous⅐NO complex, whereas O 2 has a destabilizing effect (34). Thus, we determined whether the five-coordinate species observed in single-turnover with NHA and 4-amino-BH4 originated from O 2 -induced destabilization of the six-coordinate complex.…”

The Ferrous Dioxygen Complex of the Oxygenase Domain of Neuronal Nitric-oxide Synthase

“…Both the substrate and H 4 B molecules participate in an extensive hydrogen bond network that includes a heme propionate, water molecules, and several amino acids. The proximity of the L-arginine binding site to the heme is consistent with the interactions of substrates with heme-bound ligands such as CO, NO, and imidazole, which were revealed by optical (19), EPR (20), and resonance Raman spectroscopies (21)(22)(23). In addition, the substrates and H 4 B, when present, cause a shift in the spin state of the ferric enzyme (24).…”