Thermodynamic analysis of interactions between cofactor and neuronal nitric oxide synthase

Area of Science:

• Biochemistry
• Structural Biology
• Enzyme Kinetics

Background:

• Nitric oxide synthase (NOS) is crucial for physiological processes.
• Understanding cofactor binding is key to elucidating NOS function and regulation.
• The reductase domain (Red) of nNOS plays a critical role in electron transfer.

Purpose of the Study:

• To investigate the thermodynamics of cofactor binding to the nNOS reductase domain (Red) and its mutants.
• To determine the role of different cofactor moieties in binding.
• To explore the impact of calmodulin (CaM) binding on cofactor affinity and enzyme conformation.

Main Methods:

• Isothermal titration calorimetry (ITC) was employed to study cofactor binding.
• Thermodynamic parameters (binding constant, heat capacity change) were determined.
• Deletion mutants (RedΔ40) and CaM-binding constructs (RedCaM) were analyzed.

Main Results:

• NADP(+) and 2",5 '-ADP exhibited 1:1 binding stoichiometry to Red, supporting a single-site model.
• 2 ',5 '-ADP showed significantly higher binding affinity (71 nM) and a larger negative heat capacity change (-440 cal mol(-1) K(-1)) compared to NADP+ (1.7 μM, -140 cal mol(-1) K(-1)).
• Ca(2+)/CaM binding increased 2 ',5 '-ADP affinity by 6.7-fold, primarily driven by an entropic contribution, and RedΔ40 mutant displayed reduced binding affinity.

Conclusions:

• Both adenosine and nicotinamide moieties are important for nNOS binding.
• Cofactor binding induces conformational changes in Red with distinct thermodynamic signatures.
• Ca(2+)/CaM binding promotes a more flexible 'open' conformation, facilitating electron transfer for catalysis.