Neuronal nitric oxide synthase generates superoxide from the oxygenase domain

Area of Science:

• Biochemistry
• Enzymology
• Cellular Biology

Background:

• Neuronal nitric oxide synthase (nNOS) is implicated in superoxide generation when l-arginine is depleted.
• Conflicting reports exist regarding nNOS's direct role in superoxide production, with some studies suggesting involvement of cofactors.

Purpose of the Study:

• To resolve controversies surrounding nNOS-mediated superoxide generation.
• To investigate superoxide production by wild-type nNOS and a mutant (C415A-nNOS) lacking a functional haem proximal site, without added cofactors.

Main Methods:

• Superoxide detection using a superoxide-sensitive adrenochrome assay.
• Electron spin resonance (ESR) spectroscopy with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trap.
• Assessment of nNOS mutant (C415A-nNOS) and boiled enzyme preparations.

Main Results:

• Wild-type nNOS produced significant superoxide signals, while boiled enzyme did not.
• The C415A-nNOS mutant showed a 2-fold increased lag period and markedly reduced superoxide generation (10% of wild-type).
• Sodium cyanide (NaCN) significantly inhibited superoxide formation by wild-type nNOS.

Conclusions:

• nNOS directly catalyzes superoxide formation, with the oxygenase domain being crucial.
• The reductase domain of full-length nNOS appears to be inhibited from producing superoxide.