Heme Iron Reduction and Catalysis by a Nitric Oxide Synthase Heterodimer Containing One Reductase and Two Oxygenase Domains

Siddhanta, Uma; Wu, Chaoqun; Abu-Soûd, Husam M.; Zhang, Jingli; Ghosh, Dipak; Stuehr, Dennis J.

doi:10.1074/jbc.271.13.7309

Cited by 88 publications

(95 citation statements)

References 29 publications

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“…This is similar to the results obtained by Siddhanta et al using iNOS heterodimers (15,16). They demonstrated that a single reductase domain is sufficient to support heme reduction and NO formation activity at one heme site.…”

Section: Effect Of the E592a Mutation On Electron Transfer And Dimersupporting

confidence: 91%

“…Preparation of Heterodimers-Essentially we followed the method developed by Dr. D. Stuehr (15,16). The full-length nNOSs and the oxygenase domain dimers were dissociated into monomers by incubation with 3 M urea in buffer C containing 1% Tween 20 and 2 mM ␤-ME for 1.5 h at 25°C and then for 1 h at 0°C.…”

Section: Methodsmentioning

confidence: 99%

“…CaM binding regulates electron transfer both within the reductase domain and from the reductase domain to the heme domain (11)(12)(13)(14). Siddhanta et al (15,16) reported that, in the iNOS dimer, which always binds CaM, electron transfer occurs from the flavins of one subunit to the heme domain of the other subunit during catalytic turnover. They therefore proposed a domain swap model for the structure of native iNOS.…”

mentioning

confidence: 99%

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Intra-subunit and Inter-subunit Electron Transfer in Neuronal Nitric-oxide Synthase

Sagami¹,

Daff²,

Shimizu³

2001

Journal of Biological Chemistry

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In neuronal nitric-oxide synthase (nNOS), calmodulin (CaM) binding is thought to trigger electron transfer from the reductase domain to the heme domain, which is essential for O 2 activation and NO formation. To elucidate the electron-transfer mechanism, we characterized a series of heterodimers consisting of one full-length nNOS subunit and one oxygenase-domain subunit. The results support an inter-subunit electron-transfer mechanism for the wild type nNOS, in that electrons for catalysis transfer in a Ca 2؉ /CaM-dependent way from the reductase domain of one subunit to the heme of the other subunit, as proposed for inducible NOS. This suggests that the two different isoforms form similar dimeric complexes. In a series of heterodimers containing a Ca 2؉ /CaM-insensitive mutant (delta40), electrons transferred from the reductase domain to both hemes in a Ca 2؉ /CaM-independent way. Thus, in the delta40 mutant electron transfer from the reductase domains to the heme domains can occur via both inter-subunit and intra-subunit mechanisms. However, NO formation activity was exclusively linked to inter-subunit electron transfer and was observed only in the presence of Ca 2؉ / CaM. This suggests that the mechanism of activation of nNOS by CaM is not solely dependent on the activation of electron transfer to the nNOS hemes but may involve additional structural factors linked to the catalytic action of the heme domain.

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Section: Effect Of the E592a Mutation On Electron Transfer And Dimersupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Intra-subunit and Inter-subunit Electron Transfer in Neuronal Nitric-oxide Synthase

Sagami¹,

Daff²,

Shimizu³

2001

Journal of Biological Chemistry

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“…To become active, two NOS polypeptides must form a homodimer (11)(12)(13)(14). Dimerization creates an extensive interface between two oxygenase domains, creates high affinity binding sites for (6R)-tetrahydrobiopterin (H4B) and Arg in each oxygenase domain, and enables electrons to transfer between NOS flavin and heme groups (15)(16)(17)(18)(19)(20)(21)(22)(23).…”

Section: Nitric Oxide (No)mentioning

confidence: 99%

Distinct Influence of N-terminal Elements on Neuronal Nitric-oxide Synthase Structure and Catalysis

Panda

Adak

Aulak

et al. 2003

Journal of Biological Chemistry

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Nitric oxide (NO) is a signal molecule produced in animals by three different NO synthases. Of these, only NOS I (neuronal nitric-oxide synthase; nNOS) is expressed as catalytically active N-terminally truncated forms that are missing either an N-terminal leader sequence required for protein-protein interactions or are missing the leader sequence plus three core structural motifs that in other NOS are required for dimer assembly and catalysis. To understand how the N-terminal elements impact nNOS structure-function, we generated, purified, and extensively characterized variants that were missing the N-terminal leader sequence (⌬296nNOS) or missing the leader sequence plus the three core motifs (⌬349nNOS). Eliminating the leader sequence had no impact on nNOS structure or catalysis. In contrast, additional removal of the core elements weakened but did not destroy the dimer interaction, slowed ferric heme reduction and reactivity of a hemedioxy intermediate, and caused a 10-fold poorer affinity toward substrate L-arginine. This created an nNOS variant with slower and less coupled NO synthesis that is predisposed to generate reactive oxygen species along with NO. Our findings help justify the existence of nNOS N-terminal splice variants and identify specific catalytic changes that create functional differences among them.

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“…This last step is rate-limiting (Miller et al, 1999), occurs in trans, from the NOSred of one polypeptide to the NOSox of the other. This step is triggered by conformational changes that are induced by Ca 2+ /CaM binding (Siddhanta et al, 1996).…”

Section: Ros and Nitric Oxide Synthasementioning

confidence: 99%

Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair

2007

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This review is focused on proteins with key roles in pathways controlling either reactive oxygen species or DNA damage responses, both of which are essential for preserving the nervous system. An imbalance of reactive oxygen species or inappropriate DNA damage response likely causes mutational or cytotoxic outcomes, which may lead to cancer and/or aging phenotypes. Moreover, individuals with hereditary disorders in proteins of these cellular pathways have significant neurological abnormalities. Mutations in a superoxide dismutase, which removes oxygen free radicals, may cause the neurodegenerative disease amyotrophic lateral sclerosis. Additionally, DNA repair disorders that affect the brain to varying extents include ataxia-telangiectasia-like disorder, Cockayne syndrome or Werner syndrome. Here, we highlight recent advances gained through structural biochemistry studies on enzymes linked to these disorders and other related enzymes acting within the same cellular pathways. We describe the current understanding of how these vital proteins coordinate chemical steps and integrate cellular signaling and response events. Significantly, these structural studies may provide a set of master keys to developing a unified understanding of the survival mechanisms utilized after insults by reactive oxygen species and genotoxic agents, and also provide a basis for developing an informed intervention in brain tumor and neurodegenerative disease progression.

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Heme Iron Reduction and Catalysis by a Nitric Oxide Synthase Heterodimer Containing One Reductase and Two Oxygenase Domains

Cited by 88 publications

References 29 publications

Intra-subunit and Inter-subunit Electron Transfer in Neuronal Nitric-oxide Synthase

Intra-subunit and Inter-subunit Electron Transfer in Neuronal Nitric-oxide Synthase

Distinct Influence of N-terminal Elements on Neuronal Nitric-oxide Synthase Structure and Catalysis

Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair

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