Optical and electron paramagnetic resonance absorption spectra of complexes of nitric oxide synthase I with isocyanides.
Neuronal nitric oxide synthase (NOS I) was purified from porcine brains, and optical and EPR spectra of the complexes of NOS I with isocyanides were investigated. The complex of oxidized NOS I with tert-butylisocyanide exhibited optical absorption peaks at 437 and 560 nm in the difference spectrum, whereas with tert-butylisocyanide and phenylisocyanide, optically reduced NOS I-isocyanide complexes with absorbance maxima at 433, 451, 541 and 573 nm were produced. The dissociation constants of the NOS I-isocyanide complexes were optically determined, the constants being significantly larger than those of microsomal cytochromes P-450. Phenylisocyanide did not affect the optical spectrum of oxidized NOS I. A high concentration of phenylisocyanide also had no effect on the EPR spectrum of oxidized NOS I. The optical spectra of the reduced NOS I-isocyanide complexes were pH-dependent. With increasing pH, the intensity of the absorbance at 451 nm of the complexes increased and that of the absorbance at 433 nm decreased in parallel. Upon the addition of a saturating concentration of L-arginine, the difference spectra of the reduced NOS I-phenylisocyanides complex showed a drastic change, i.e., an increase in optical intensity at 433 nm and a concomitant decrease in the intensity at 451 nm. In titration experiments with L-arginine, spectral binding, Ks = 2.5 microM, was determined from the absorbance increase at 433 nm. No spectral change was observed on the addition of the same concentration of D-arginine. N(omega)-nitro-L-arginine methyl ester (NAME), a potent inhibitor of NOS I, had a similar effect to L-arginine, but the time course of the spectral change was very slow. These results suggest that: (1) the heme-iron pocket of NOS I will be narrower than those of the microsomal and mitochondrial cytochromes P-450; and (2) the binding of L-arginine and its analogue to their binding sites caused conformational changes around the ferrous heme moiety of NOS I.
Yoneyama
H
H
Department of Biochemistry, School of Medicine, Kagawa Medical University, Kita-gun, Japan.
Hori
H
H
Ichikawa
Y
Y
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D003486:Cyanides / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008862:Microsomes, Liver / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011817:Rabbits; D013053:Spectrophotometry; D013552:Swine
D003486:Cyanides; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D019001:Nitric Oxide Synthase