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.