Oxygen activation in neuronal NO synthase: resolving the consecutive mono-oxygenation steps

Area of Science:

• Biochemistry
• Enzymology
• Chemical Biology

Background:

• Mammalian nitric oxide synthase (NOS) enzymes catalyze the two-step conversion of L-arginine to nitric oxide (NO) and citrulline.
• Both steps are believed to involve cofactor tetrahydrobiopterin (BH4)-mediated activation of dioxygen at the enzyme's heme site, but reaction intermediates remain poorly characterized.

Purpose of the Study:

• To investigate the distinct chemical mechanisms of the two mono-oxygenation steps in NO synthesis.
• To characterize novel heme intermediates formed during NOS catalysis using a modified enzyme.

Main Methods:

• Utilized the G586S mutant of neuronal NOS (nNOS), engineered with an additional active site hydrogen bond and proton source.
• Analyzed the catalytic activity and intermediate formation of the mutant enzyme in the presence of L-arginine, Nω-hydroxy-L-arginine (NOHA), and tetrahydrobiopterin (BH4).
• Spectroscopic methods were employed to detect and characterize transient heme species.

Main Results:

• The G586S nNOS mutant successfully activated dioxygen with BH4, similar to wild-type, and formed a novel, stabilized intermediate heme species.
• The mutant enzyme catalyzed the second step (NOHA to NO conversion) but failed to perform the first step (L-arginine to NOHA conversion).
• This functional difference indicates the mutant enzyme can discriminate between the two mono-oxygenation steps.

Conclusions:

• The two sequential mono-oxygenation steps in NOS catalysis proceed via distinct chemical mechanisms.
• The engineered hydrogen bond in the G586S nNOS mutant stabilizes a key oxygenating species, providing insight into the reaction pathway.
• This study provides evidence for mechanistic divergence in NO synthesis, challenging previous assumptions of identical reaction pathways.