Neuronal nitric-oxide synthase interaction with calmodulin-troponin C chimeras.
Calmodulin (CaM) binding activates neuronal nitric-oxide synthase (nNOS) catalytic functions and also up-regulates electron transfer into its flavin and heme centers. Here, we utilized seven tight binding CaM-troponin C chimeras, which variably activate nNOS NO synthesis to examine the relationship between CaM domain structure, activation of catalytic functions, and control of internal electron transfer at two points within nNOS. Chimeras that were singly substituted with troponin C domains 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 synthesis was approximately proportional to the rate of heme iron reduction supported by each chimera, which varied from 0% to approximately 80% compared with native CaM and remained coupled to NO synthesis in all cases. In contrast, chimera activation of cytochrome c reduction was not always associated with accelerated reduction of nNOS flavins, and certain chimeras activated cytochrome c reduction without triggering heme iron 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 activity by two mechanisms, only one of which is associated with an increased rate of flavin reduction.
Gachhui
R
R
Department of Immunology, The Cleveland Clinic Research Institute, Cleveland, Ohio 44195, USA.
Abu-Soud
H M
HM
Ghosha
D K
DK
Presta
A
A
Blazing
M A
MA
Mayer
B
B
George
S E
SE
Stuehr
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000502:physiology; D002147:Calmodulin / Q000737:chemistry / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D005420:Flavoproteins / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000494:pharmacology; D016415:Sequence Alignment; D019209:Troponin C / Q000737:chemistry / Q000494:pharmacology
D002147:Calmodulin; D003574:Cytochrome c Group; D005420:Flavoproteins; D011993:Recombinant Fusion Proteins; D019209:Troponin C; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D019001:Nitric Oxide Synthase