Electron Paramagnetic Resonance Studies on Substrate Binding to the NO Complex of Neuronal Nitric Oxide Synthase

Summary

The nitric oxide synthases (NOS), a group of flavo-heme enzymes, biosynthesize a physiologically versatile diatomic messenger, nitric oxide (NO), from the substrate l-arginine (l-Arg). We have examined the active site of the neuronal isoform by electron paramagnetic resonance (EPR) spectroscopy of the ferrous NO complex. The substrate-free NO-bound enzyme exhibits a cytochrome P-450-type EPR spectrum of a typical hexa-coordinate NO heme complex with a nonnitrogenous proximal axial heme ligand. The substrate-free NO complex is rather unstable and spontaneously converts to a cytochrome P-420-type penta-coordinate denatured form. Binding of l-Arg enhances the stability of the hexa-coordinate NO form. The EPR spectrum of the NO adduct of the enzyme-arginine complex has an increased g-anisotropy and well-resolved hyperfine coupling as the result of the ¹⁴N of nitric oxide. Significant changes in the NO EPR spectrum were observed on N^ω-hydroxy-l-Arg (NHA) binding. These changes are strong indications of a direct interaction between the l-Arg and the bound NO in the distal heme pocket of the enzyme. Electrostatic interactions between the guanidino group of arginine and the bound NO or the steric effect of the substrates appears to affect the Fe-NO geometry, resulting in the observed spectral changes.