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J. Biol. Chem., Vol. 275, Issue 38, 29225-29232, September 22, 2000
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From the 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.
The C Termini of Constitutive Nitric-oxide Synthases Control
Electron Flow through the Flavin and Heme Domains and Affect Modulation
by Calmodulin*
§,,,,,,
Department of Biochemistry, The University
of Texas Health Science Center, San Antonio, Texas 78229 and the
¶ Department of Biochemistry, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
*
This work was supported by National Institutes of Health
Grant GM52419, Robert A. Welch Foundation Grant AQ1192 (to
B. S. S. M.), and National Institutes of Health Grant GM52682 (to
J.-J. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence may be addressed: Dept. of
Biochemistry, the University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229. Tel.: 210-567-6627; Fax: 210-567-6984; E-mail: masters@uthscsa.edu.
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