Crucial Role of Lys423 in the Electron Transfer of Neuronal Nitric-oxide Synthase

Nitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H 4 B) cofactor. During Arg hydroxylation, H 4 B acts as a one-electron donor and is then presumed to redox cycle (i.e. be reduced back to H 4 B) within NOS before further catalysis can proceed. Whereas H 4 B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H 4 B, and a method that could separately quantify the flavin and pterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical following Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H 4 B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a "throughheme" mechanism may operate for H 4 B radical reduction in NOS.

Section: Discussionmentioning

confidence: 99%

Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase

Wei

Wang

Tejero

et al. 2008

“…Full-length nNOS wild-type and mutants (F584L, W587L, and RedCaM) were purified with DEAEToyopearl 650 M (Tosoh Co., Tokyo, Japan), 2Ј,5Ј-ADP-Sepharose and calmodulin-Sepharose column chromatography, as previously described (16,33,34). To avoid denaturation of the mutants, we used a buffer containing 5 M H 4 B.…”

Section: Methodsmentioning

confidence: 99%

“…Expression and Preparation of Full-length Wild-type nNOS and the nNOS Mutants-Full-length nNOS wild-type enzyme and the mutants were expressed in the E. coli cell line BL21 (DE3), which contains plasmid pGroESL for expression of chaperone proteins, as previously described (16,33,34). Full-length nNOS wild-type and mutants (F584L, W587L, and RedCaM) were purified with DEAEToyopearl 650 M (Tosoh Co., Tokyo, Japan), 2Ј,5Ј-ADP-Sepharose and calmodulin-Sepharose column chromatography, as previously described (16,33,34).…”

Section: Methodsmentioning

confidence: 99%

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

Sato

Sagami

Shimizu

2004

Self Cite

Caveolin is known to down-regulate both neuronal (nNOS) and endothelial nitric-oxide synthase (eNOS). In the present study, direct interactions of recombinant caveolin-1 with both the oxygenase and reductase domains of nNOS were demonstrated using in vitro binding assays. To elucidate the mechanism of nNOS regulation by caveolin, we examined the effects of a caveolin-1 scaffolding domain peptide (CaV1p1; residues (82-101)) on the catalytic activities of wild-type and mutant nNOSs. CaV1p1 inhibited NO formation activity and NADPH oxidation of wild-type nNOS in a dose-dependent manner with an IC 50 value of 1.8 M. Mutations of Phe 584 and Trp 587 within a caveolin binding consensus motif of the oxygenase domain did not result in the loss of CaV1p1 inhibition, indicating that an alternate region of nNOS mediates inhibition by caveolin. The addition of CaV1p1 also inhibited more than 90% of the cytochrome c reductase activity in the isolated reductase domain with or without the calmodulin (CaM) binding site, whereas CaV1p1 inhibited ferricyanide reductase activity by only 50%. These results suggest that there are significant differences in the mechanism of inhibition by caveolin for nNOS as compared with those previously reported for eNOS. Further analysis of the interaction through the use of several reductase domain deletion mutants revealed that the FMN domain was essential for successful interaction between caveolin-1 and nNOS reductase. We also examined the effects of CaV1p1 on an autoinhibitory domain deletion mutant (⌬40) and a C-terminal truncation mutant (⌬C33), both of which are able to form NO in the absence of CaM, unlike the wild-type enzyme. Interestingly, CaV1p1 inhibited CaM-dependent, but not CaMindependent, NO formation activities of both ⌬40 and ⌬C33, suggesting that CaV1p1 inhibits interdomain electron transfer induced by CaM from the reductase domain to the oxygenase domain.Nitric-oxide synthase (NOS) 1 generates the physiologically important nitric oxide molecule (NO) from L-arginine (L-Arg) (1-5).NOS consists of two functional domains: an N-terminal oxygenase domain containing a cytochrome P450 (P450)-like heme active site, and L-Arg and (6R)-5,6,7,8-tetrahydrobiopterin (H 4 B) binding sites (6), and a C-terminal reductase domain that contains the FAD, FMN, and NADPH binding sites (7,8). The two domains are connected by a calmodulin (CaM) binding site. Binding of CaM facilitates the interdomain electron transfers required for NO formation as well as intradomain electron transfers within the reductase domain. Two isoforms of NOS (neuronal NOS (nNOS) and endothelial NOS (eNOS)) are constitutively expressed and are regulated by intracellular Ca 2ϩ concentrations via the reversible binding of Ca 2ϩ /CaM (9). In contrast, the inducible NOS isoform (iNOS) is regulated at the transcriptional level, and binds CaM tightly even at basal Ca 2ϩ concentrations (10). CaM-dependent regulation of the three NOS isoforms is also controlled by isoform-specific sequences (i.e. insertions or extensions) and by pho...

“…described in the tables are low compared with the relevant NO formation rates and the NADPH oxidation rates as reported for the wild type homodimer in our previous studies (20,22). The heme reduction described here was determined at 15°C under anaerobic conditions, whereas NADPH consumption and NO formation rates were determined at 25°C under aerobic conditions.…”

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

confidence: 99%

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

Sagami¹,

Daff²,

Shimizu³

2001

Self Cite

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.