Substrate- and isoform-specific dioxygen complexes of nitric oxide synthase

Area of Science:

• Biochemistry
• Enzymology
• Spectroscopy

Background:

• Nitric oxide synthase (NOS) produces nitric oxide (NO) through a two-step reaction.
• The first step converts L-arginine (Arg) to N-hydroxy-L-arginine (NOHA), and the second converts NOHA to citrulline and NO.
• Understanding the mechanistic differences between these steps is crucial for enzyme function.

Purpose of the Study:

• To investigate the mechanistic differences in the two catalytic steps of NOS.
• To study the structural properties of dioxygen-bound complexes of the inducible NOS oxygenase domain (iNOSoxy).

Main Methods:

• Resonance Raman spectroscopy.
• A homemade continuous-flow rapid solution mixer.
• Studied substrate-free and substrate-bound iNOSoxy complexes.

Main Results:

• Identified an O-O stretching frequency of 1133 cm-1 in substrate-free iNOSoxy.
• Observed a shift to 1126 cm-1 upon L-arginine binding, indicating H-bonding to the heme iron-bound dioxygen.
• No shift in O-O frequency was observed upon NOHA binding, suggesting a lack of direct H-bonding.

Conclusions:

• Substrate-specific hydrogen bonding interactions are critical for NOS catalysis.
• H-bonds from L-arginine facilitate O-O bond cleavage for NO formation.
• The absence of H-bonds during NOHA binding prevents premature O-O bond cleavage, allowing for nucleophilic addition.