Domain swapping in inducible nitric-oxide synthase

Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer

Uma Siddhanta, Anthony Presta, Baochen Fan, Dennis Wolan, Denis L. Rousseau, Dennis J. Stuehr

Research output: Contribution to journalArticle

178 Citations (Scopus)

Abstract

Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and L-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis from L-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem, 271, 7309-7312). Here, we characterize a pair of heterodimers that contain an L-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented L-Arg binding to one oxygenase domain in each heterodimer but did not affect the L-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-L-Arg interaction confirmed that E371A only prevents L-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.

Original languageEnglish (US)
Pages (from-to)18950-18958
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number30
DOIs
StatePublished - Jul 24 1998
Externally publishedYes

Fingerprint

Oxygenases
Nitric Oxide Synthase Type II
Heme
Dimers
Electrons
Oxidoreductases
Nitric Oxide
Mutation
Arginine
Iron
Flavins
4,6-dinitro-o-cresol
Flavin Mononucleotide
Flavin-Adenine Dinucleotide
Dimerization
Calmodulin
NADP
Cytokines

ASJC Scopus subject areas

  • Biochemistry

Cite this

Domain swapping in inducible nitric-oxide synthase : Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer. / Siddhanta, Uma; Presta, Anthony; Fan, Baochen; Wolan, Dennis; Rousseau, Denis L.; Stuehr, Dennis J.

In: Journal of Biological Chemistry, Vol. 273, No. 30, 24.07.1998, p. 18950-18958.

Research output: Contribution to journalArticle

Siddhanta, Uma ; Presta, Anthony ; Fan, Baochen ; Wolan, Dennis ; Rousseau, Denis L. ; Stuehr, Dennis J. / Domain swapping in inducible nitric-oxide synthase : Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 30. pp. 18950-18958.
@article{5c9cc5e9d0fc401db64b8baca52d5662,
title = "Domain swapping in inducible nitric-oxide synthase: Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer",
abstract = "Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and L-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis from L-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem, 271, 7309-7312). Here, we characterize a pair of heterodimers that contain an L-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented L-Arg binding to one oxygenase domain in each heterodimer but did not affect the L-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-L-Arg interaction confirmed that E371A only prevents L-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.",
author = "Uma Siddhanta and Anthony Presta and Baochen Fan and Dennis Wolan and Rousseau, {Denis L.} and Stuehr, {Dennis J.}",
year = "1998",
month = "7",
day = "24",
doi = "10.1074/jbc.273.30.18950",
language = "English (US)",
volume = "273",
pages = "18950--18958",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "30",

}

TY - JOUR

T1 - Domain swapping in inducible nitric-oxide synthase

T2 - Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer

AU - Siddhanta, Uma

AU - Presta, Anthony

AU - Fan, Baochen

AU - Wolan, Dennis

AU - Rousseau, Denis L.

AU - Stuehr, Dennis J.

PY - 1998/7/24

Y1 - 1998/7/24

N2 - Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and L-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis from L-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem, 271, 7309-7312). Here, we characterize a pair of heterodimers that contain an L-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented L-Arg binding to one oxygenase domain in each heterodimer but did not affect the L-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-L-Arg interaction confirmed that E371A only prevents L-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.

AB - Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and L-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis from L-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem, 271, 7309-7312). Here, we characterize a pair of heterodimers that contain an L-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented L-Arg binding to one oxygenase domain in each heterodimer but did not affect the L-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-L-Arg interaction confirmed that E371A only prevents L-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.

UR - http://www.scopus.com/inward/record.url?scp=0032563106&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032563106&partnerID=8YFLogxK

U2 - 10.1074/jbc.273.30.18950

DO - 10.1074/jbc.273.30.18950

M3 - Article

VL - 273

SP - 18950

EP - 18958

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 30

ER -