NEURONAL NITRIC-OXIDE SYNTHASE INTERACTION WITH CALMODULIN-TROPONIN-CCHIMERAS

Calmodulin (CaM) binding activates neuronal nitric-oxide synthase (nNO S) catalytic functions and also upregulates electron transfer into its flavin and heme centers, Here, we utilized seven tight binding CaM-tr oponin C chimeras, which variably activate nNOS NO synthesis to examin e the relationship between CaM domain structure, activation of catalyt ic functions, and control of internal electron transfer at two points within nNOS, Chimeras that were singly substituted with troponin C dom ains 4, 3, 2, or 1 were increasingly unable to activate NO synthesis, but all caused some activation of cytochrome c reduction compared with CaM-free nNOS, The magnitude by which each chimera activated NO synth esis was approximately proportional to the rate of heme iron reduction supported by each chimera, which varied from 0% to similar to 80% com pared with native CaM and remained coupled to NO synthesis in all case s, In contrast, chimera activation of cytochrome c reduction was not a lways associated with accelerated reduction of nNOS flavins, and certa in chimeras activated cytochrome c reduction without triggering heme i ron reduction, We conclude: 1) CaM effects on electron transfer at two points within nNOS can be functionally separated. 2) CaM controls NO synthesis by governing heme iron reduction, but enhances reductase act ivity by two mechanisms, only one of which is associated with an incre ased rate of flavin reduction.