Optical and electron paramagnetic resonance absorption spectra of complexes of nitric oxide synthase I with isocyanides

• Published in: Biochimica et biophysica acta Vol. 1335; no. 3; pp. 253 - 264
• Main Authors: Yoneyama, Hirohito, Hori, Hiroshi, Ichikawa, Yoshiyuki
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 06.06.1997
• Subjects: Animals; Binding Sites; Brain - enzymology; Cyanides - chemistry; Cytochrome P-450; Cytochrome P-450 Enzyme System - chemistry; Electron Spin Resonance Spectroscopy; Heme - chemistry; Hydrogen-Ion Concentration; Microsomes, Liver - enzymology; Monooxygenase; Nitric oxide synthase; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase - isolation & purification; Nitric oxide synthase I-isocyanide complex; Oxidation-Reduction; Phenylisocyanide; Rabbits; Spectrophotometry; Swine; EDTA, ethylenediamine tetraacetate dihydrate; Phenylisocyanide; NOS I, neuronal nitric oxide synthase; Nitric oxide synthase I-isocyanide complex; EPR, electron paramagnetic resonance; MOPS, 3-[ N-morpholino]propanesulfonic acid; Cytochrome P-450; Nitric oxide synthase; NOS III, endothelial nitric oxide synthase; NOS, nitric oxide synthase; Monooxygenase; NAME, N ω -nitro- l-arginine methyl ester
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(96)00142-0

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, K s=2.5 μM, 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 ω -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.