Calmodulin activates intersubunit electron transfer in the neuronal nitric-oxide synthase dimer

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Neuronal nitric oxide synthase (nNOS) is a complex enzyme with distinct oxygenase and reductase domains.
• nNOS activity depends on dimerization and the binding of cofactors like heme, tetrahydrobiopterin, and NADPH.
• Calmodulin binding is known to regulate nNOS activity, but its precise role in electron transfer is not fully understood.

Purpose of the Study:

• To elucidate the mechanism of calmodulin-induced electron transfer in nNOS dimers.
• To investigate the role of specific domains and mutations in nNOS activity and calmodulin interaction.
• To determine how calmodulin binding influences electron transfer between the reductase and oxygenase domains.

Main Methods:

• Construction and analysis of four distinct nNOS heterodimers using wild-type and mutant subunits (G671A, E592A).
• Assessment of Arg binding, NO synthesis, and catalytic responses to calmodulin variants.
• Utilizing predominantly monomeric and Arg-binding mutants to dissect domain interactions.

Main Results:

• The E592A mutation impaired Arg binding and NO synthesis when in the oxygenase domain adjacent to the full-length subunit.
• An active heterodimer, comprising a G671A/E592A full-length subunit and a wild-type oxygenase domain, mimicked wild-type nNOS activity.
• Catalytic responses to various calmodulin forms were similar between the active heterodimer and wild-type homodimeric nNOS.

Conclusions:

• Calmodulin actively supports trans-electron transfer between flavin and heme groups on adjacent subunits in nNOS dimers.
• Calmodulin's function involves aligning the reductase and oxygenase domains within a nNOS dimer to facilitate electron transfer.
• Proper domain alignment by calmodulin is essential for initiating heme-mediated NO synthesis.