A conserved aspartate (Asp-1393) regulates NADPH reduction of neuronal nitric-oxide synthase: Implications for catalysis

Koustubh Panda, Subrata Adak, David Konas, Manisha Sharma, Dennis J. Stuehr

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)18323-18333
Number of pages11
JournalJournal of Biological Chemistry
Volume279
Issue number18
DOIs
StatePublished - 30 Apr 2004

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Nitric Oxide Synthase Type I
Calmodulin
Catalysis
NADP
Aspartic Acid
Heme
Flavoproteins
Ferredoxin-NADP Reductase
Flavin-Adenine Dinucleotide
Nitric Oxide Synthase
Flavins
Electrons
NADPH-Ferrihemoprotein Reductase
NAD
Binding Sites
Cytochromes c
Catalyst activity
Enzymes
Defects
4,6-dinitro-o-cresol

Cite this

Panda, Koustubh ; Adak, Subrata ; Konas, David ; Sharma, Manisha ; Stuehr, Dennis J. / A conserved aspartate (Asp-1393) regulates NADPH reduction of neuronal nitric-oxide synthase : Implications for catalysis. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 18. pp. 18323-18333.
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A conserved aspartate (Asp-1393) regulates NADPH reduction of neuronal nitric-oxide synthase : Implications for catalysis. / Panda, Koustubh; Adak, Subrata; Konas, David; Sharma, Manisha; Stuehr, Dennis J.

In: Journal of Biological Chemistry, Vol. 279, No. 18, 30.04.2004, p. 18323-18333.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A conserved aspartate (Asp-1393) regulates NADPH reduction of neuronal nitric-oxide synthase

T2 - Implications for catalysis

AU - Panda, Koustubh

AU - Adak, Subrata

AU - Konas, David

AU - Sharma, Manisha

AU - Stuehr, Dennis J.

PY - 2004/4/30

Y1 - 2004/4/30

N2 - 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.

AB - 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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DO - 10.1074/jbc.M310391200

M3 - Article

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AN - SCOPUS:2442435820

VL - 279

SP - 18323

EP - 18333

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

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