SUBSTRATE BINDING-INDUCED CHANGES IN THE EPR-SPECTRA OF THE FERROUS NITRIC-OXIDE COMPLEXES OF NEURONAL NITRIC-OXIDE SYNTHASE

A versatile diatomic physiological messenger, nitric oxide (NO), is bi osynthesized by a group of flavo-heme enzymes, the nitric oxide syntha ses. We have examined the active site of the neuronal isoform by EPR s pectroscopy of the ferrous nitric oxide complex. The nitric oxide comp lex of the substrate-free enzyme exhibits a cytochrome P450-type EPR s pectrum typical of a hexacoordinate NO-heme complex with a non-nitroge nous proximal axial heme ligand. The NO complex of the substrate-free enzyme is rather unstable and spontaneously converts to a cytochrome P 420 type pentacoordinate denatured form. Binding of L-arginine (L-Arg) enhances the stability of the hexacoordinate NO form. The EPR spectru m of the NO adduct of the enzyme-substrate complex has an increased g- anisotropy and well-resolved hyperfine couplings due to the N-14 of ni tric oxide. Significant perturbations in the NO EPR spectrum were obse rved upon N-omega-monomethyl-L-Arg and N-omega-hydroxy-L-Arg binding. The perturbations in the EPR spectrum indicate that L-Arg and its deri vatives bind on the distal site of the heme in very close proximity to the bound NO to cause alterations in the heme-NO coordination structu re. Interactions between the bound NO and the substrate or its analogu es appear to affect the Fe-NO geometry, resulting in the observed spec tral changes. We infer that analogous interactions with oxygen might b e involved in the hydroxylation events during enzyme catalysis of nitr ic oxide synthase.