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J. Biol. Chem., Vol. 276, Issue 2, 1244-1252, January 12, 2001
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From the Rat neuronal NO synthase (nNOS) contains an
Akt-dependent phosphorylation motif in its reductase
domain. We mutated a target residue in that site (Ser-1412 to Asp) to
mimic phosphorylation and then characterized the mutant using
conventional and stopped-flow spectroscopies. Compared with wild-type,
S1412D nNOS catalyzed faster cytochrome c and ferricyanide
reduction but displayed slower steady-state NO synthesis with greater
uncoupling of NADPH oxidation. Paradoxically, the mutant had faster
heme reduction, faster heme-NO complex formation, and greater heme-NO
complex accumulation at steady state. To understand how these behaviors
related to flavin and heme reduction rates, we utilized three soybean
calmodulins (CaMs) that supported a range of slower flavin and heme
reduction rates in mutant and wild-type nNOS. Reductase activity and
two catalytic parameters (speed and amount of heme-NO complex
formation) related directly to the speed of flavin and heme reduction.
In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We
conclude the following. 1) The S1412D mutation speeds electron transfer
out of the reductase domain. 2) Faster heme reduction speeds intrinsic
NO synthesis but diminishes NO release in the steady state. 3) Heme
reduction displays an optimum regarding NO release during steady state.
The unique behavior of S1412D nNOS reveals the importance of heme
reduction rate in controlling steady-state activity and suggests that
nNOS already has a near-optimal rate of heme reduction.
Neuronal Nitric-oxide Synthase Mutant (Ser-1412
Asp) Demonstrates Surprising Connections between Heme Reduction, NO
Complex Formation, and Catalysis*
§,,,
Department of Immunology, Lerner Research
Institute, Cleveland Clinic, Cleveland, Ohio 44195 and the
¶ Department of Medical Biochemistry, Ohio State University,
Columbus, Ohio 43210
*
This work was supported by National Institutes of Health
Grants GM51491 (to D. J. S.) and DK33727 (to D. J).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: Immunology NB-3,
Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave.,
Cleveland, OH 44195. Tel.: 216-445-6950; Fax: 216-444-9329; E-mail:
stuehrd@ccf.org.
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