Ca²⁺-independent activity of nitric oxide synthase

Ca²⁺-independent forms of nitric-oxide synthase have significant activity when the endogenous calmodulin subunit is Ca²⁺ free. Further activation is seen when Ca²⁺ is added. We have examined the activation of a Ca²⁺-independent nitric-oxide synthase variant and its two point mutants that are more dependent on Ca²⁺ for activation using mutant calmodulins containing non-functional Ca²⁺-binding sites. These studies provide evidence that the Ca²⁺-independent activity of these enzymes can be exerted through specific adapted interactions between the enzyme and the Ca²⁺-binding site 2 of calmodulin. Further, the results suggest that EGTA-sensitive metals other than Ca²⁺ complexed to calmodulin may be involved in maximal activation of these nitric-oxide synthase variants.