The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin.
The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20-40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.
Roman
L J
LJ
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229, USA. roman@uthscsa.edu
Martásek
P
P
Miller
R T
RT
Harris
D E
DE
de La Garza
M A
MA
Shea
T M
TM
Kim
J J
JJ
Masters
B S
BS
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D051381:Rats; D016415:Sequence Alignment
D002147:Calmodulin; D005415:Flavins; D006418:Heme; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat