A Conserved Aspartate (Asp-1393) Regulates NADPH Reduction of Neuronal Nitric-oxide Synthase
Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM). The NOS flavoprotein domain includes a ferredoxin-NADP + reductase (FNR)-like module that contains NADPH- and FAD-binding sites. FNR-like modules in related flavoproteins have t...
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Published in | The Journal of biological chemistry Vol. 279; no. 18; pp. 18323 - 18333 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
American Society for Biochemistry and Molecular Biology
01.04.2004
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Online Access | Get full text |
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Summary: | Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM).
The NOS flavoprotein domain includes a ferredoxin-NADP + reductase (FNR)-like module that contains NADPH- and FAD-binding sites. FNR-like modules in related flavoproteins have three
conserved residues that regulate electron transfer between bound NAD(P)H and FAD. To investigate the function of one of these
residues in neuronal NOS (nNOS), we generated and characterized mutants that had Val, Glu, or Asn substituted for the conserved
Asp-1393. All three mutants exhibited normal composition, spectral properties, and binding of cofactors, substrates, and CaM.
All had slower NADPH-dependent cytochrome c and ferricyanide reductase activities, which were associated with proportionally slower rates of NADPH-dependent flavin reduction
in the CaM-free and CaM-bound states. Rates of NO synthesis were also proportionally slower in the mutants and were associated
with slower rates of CaM-dependent ferric heme reduction. However, a D1393V mutant whose flavins had been prereduced with
NADPH had a normal rate of heme reduction. This indicated that the kinetic defect was restricted to flavin reduction step(s)
in the mutants and suggested that this limited their catalytic activities. Together, our results show the following. 1) The
presence and positioning of the Asp-1393 carboxylate side chain are critical to enable NADPH-dependent reduction of the nNOS
flavoprotein. 2) Control of flavin reduction is important because it ensures that the rate of heme reduction is sufficiently
fast to enable NO synthesis by nNOS. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M310391200 |