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J. Biol. Chem., Vol. 276, Issue 26, 23246-23252, June 29, 2001
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From the Department of Immunology, Lerner Research
Institute, Cleveland Clinic, Cleveland, Ohio 44195
Neuronal nitric-oxide synthase (nNOS or NOS
I) and endothelial NOS (eNOS or NOS III) differ widely in their
reductase and nitric oxide (NO) synthesis activities, electron transfer
rates, and propensities to form a heme-NO complex during catalysis. We generated chimeras by swapping eNOS and nNOS oxygenase domains to understand the basis for these differences and to identify structural elements that determine their catalytic behaviors. Swapping
oxygenase domains did not alter domain-specific catalytic functions
(cytochrome c reduction or
H2O2-supported
N
Chimeras of Nitric-oxide Synthase Types I and III Establish
Fundamental Correlates between Heme Reduction, Heme-NO Complex
Formation, and Catalytic Activity*
,
-hydroxy-L-arginine oxidation)
but markedly affected steady-state NO synthesis and NADPH oxidation
compared with native eNOS and nNOS. Stopped-flow analysis showed that
reductase domains either maintained (nNOS) or slightly exceeded
(eNOS) their native rates of heme reduction in each chimera. Heme
reduction rates were found to correlate with the initial rates of NADPH
oxidation and heme-NO complex formation, with the percentage of heme-NO
complex attained during the steady state, and with NO synthesis
activity. Oxygenase domain identity influenced these parameters to a
lesser degree. We conclude: 1) Heme reduction rates in nNOS and eNOS
are controlled primarily by their reductase domains and are almost
independent of oxygenase domain identity. 2) Heme reduction rate is the
dominant parameter controlling the kinetics and extent of heme-NO
complex formation in both eNOS and nNOS, and thus it determines to what degree heme-NO complex formation influences their steady-state NO
synthesis, whereas oxygenase domains provide minor but important influences. 3) General principles that relate heme reduction
rate, heme-NO complex formation, and NO synthesis are not specific for nNOS but apply to eNOS as well.
*
This work was supported by National Institutes of Health
Grant GM51491 (to D. J. S.) and a fellowship from the American Heart Association (to S. A.).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 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 or adaks{at}ccf.org.
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