Abstract
Nitric oxide (NO) synthases (NOS) are thiolate-ligated heme-, tetrahydrobiopterin (BH4)-, and flavin-containing monooxygenases which catalyze the NADPH-dependent conversion of L-arginine (L-Arg) to NO and citrulline. NOS consists of two domians: an N-terminal oxygenase (heme- and BH4-bound) domain and a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we have spectroscopically examined the binding of L-Arg and BH4 to the dimeric, BH4-free ferric neuronal NOS (nNOS) oxygenase domain expressed in Escherichia coli separately from the reductase domain. Addition of L-Arg or its analogue inhibitors (N(G)-methyl-L-Arg, N(G)-nitro-L-Arg) and BH4, together with dithiothreitol (DTT), to the pterin-free ferric low-spin oxygenase domain (λ(max): 419,538, 568 nm) and incubation for 2-3 days at 4 °C converted the domain to a native enzyme-type, predominantly high-spin state (λ(max): ~395, ~512, ~650 nm). 7,8-Dihydrobiopterin and other thiols (e.g., β-mercaptoethanol, cysteine, and glutathione, with less effectiveness) can replace BH4 and DTT, respectively. The UV-visible absorption spectrum of L-Arg-bound ferric full-length nNOS, which exhibits a relatively intense band at ~650 nm (ε = 7.5-8 mM-1 cm-1) due to the presence of a neutral flavin semiquinone, can then be quantitatively reconstructed by combining the spectra of equimolar amounts of the oxygenase and reductase domains. Of particular note, the heme spin-state conversion does not occur in the absence of a thiol even after prolonged (35-48 h) incubation of the oxygenase domain with BH4 and/or L-Arg under anaerobic conditions. Thus, DTT (or other thiols) plays a significant role(s) beyond keeping BH4 in its reduced form, in restoring the pterin- and/or substrate- binding capability of the E. coli-expressed, BH4-free, dimeric nNOS oxygenase domain. Our results in combination with recently available X-ray crystallography and site-directed mutagenesis data suggest that the observed DTT effects arise from the involvement of an intersubunit disulfide bond or its rearrangement in the NOS dimer.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 15853-15862 |
| Number of pages | 10 |
| Journal | Biochemistry |
| Volume | 38 |
| Issue number | 48 |
| DOIs | |
| State | Published - Nov 30 1999 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Essential thiol requirement to restore pterin- or substrate-binding capability and to regenerate native enzyme-type high-spin heme spectra in the Escherichia coli-expressed tetrahydrobiopterin-free oxygenase domain of neuronal nitric oxide synthase. / Sono, Masanori; Ledbetter, Amy P.; McMillan, Kirk; Roman, Linda J.; Shea, Thomas M.; Masters, Bettie Sue Siler; Dawson, John H.
In: Biochemistry, Vol. 38, No. 48, 30.11.1999, p. 15853-15862.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Essential thiol requirement to restore pterin- or substrate-binding capability and to regenerate native enzyme-type high-spin heme spectra in the Escherichia coli-expressed tetrahydrobiopterin-free oxygenase domain of neuronal nitric oxide synthase
AU - Sono, Masanori
AU - Ledbetter, Amy P.
AU - McMillan, Kirk
AU - Roman, Linda J.
AU - Shea, Thomas M.
AU - Masters, Bettie Sue Siler
AU - Dawson, John H.
PY - 1999/11/30
Y1 - 1999/11/30
N2 - Nitric oxide (NO) synthases (NOS) are thiolate-ligated heme-, tetrahydrobiopterin (BH4)-, and flavin-containing monooxygenases which catalyze the NADPH-dependent conversion of L-arginine (L-Arg) to NO and citrulline. NOS consists of two domians: an N-terminal oxygenase (heme- and BH4-bound) domain and a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we have spectroscopically examined the binding of L-Arg and BH4 to the dimeric, BH4-free ferric neuronal NOS (nNOS) oxygenase domain expressed in Escherichia coli separately from the reductase domain. Addition of L-Arg or its analogue inhibitors (N(G)-methyl-L-Arg, N(G)-nitro-L-Arg) and BH4, together with dithiothreitol (DTT), to the pterin-free ferric low-spin oxygenase domain (λ(max): 419,538, 568 nm) and incubation for 2-3 days at 4 °C converted the domain to a native enzyme-type, predominantly high-spin state (λ(max): ~395, ~512, ~650 nm). 7,8-Dihydrobiopterin and other thiols (e.g., β-mercaptoethanol, cysteine, and glutathione, with less effectiveness) can replace BH4 and DTT, respectively. The UV-visible absorption spectrum of L-Arg-bound ferric full-length nNOS, which exhibits a relatively intense band at ~650 nm (ε = 7.5-8 mM-1 cm-1) due to the presence of a neutral flavin semiquinone, can then be quantitatively reconstructed by combining the spectra of equimolar amounts of the oxygenase and reductase domains. Of particular note, the heme spin-state conversion does not occur in the absence of a thiol even after prolonged (35-48 h) incubation of the oxygenase domain with BH4 and/or L-Arg under anaerobic conditions. Thus, DTT (or other thiols) plays a significant role(s) beyond keeping BH4 in its reduced form, in restoring the pterin- and/or substrate- binding capability of the E. coli-expressed, BH4-free, dimeric nNOS oxygenase domain. Our results in combination with recently available X-ray crystallography and site-directed mutagenesis data suggest that the observed DTT effects arise from the involvement of an intersubunit disulfide bond or its rearrangement in the NOS dimer.
AB - Nitric oxide (NO) synthases (NOS) are thiolate-ligated heme-, tetrahydrobiopterin (BH4)-, and flavin-containing monooxygenases which catalyze the NADPH-dependent conversion of L-arginine (L-Arg) to NO and citrulline. NOS consists of two domians: an N-terminal oxygenase (heme- and BH4-bound) domain and a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we have spectroscopically examined the binding of L-Arg and BH4 to the dimeric, BH4-free ferric neuronal NOS (nNOS) oxygenase domain expressed in Escherichia coli separately from the reductase domain. Addition of L-Arg or its analogue inhibitors (N(G)-methyl-L-Arg, N(G)-nitro-L-Arg) and BH4, together with dithiothreitol (DTT), to the pterin-free ferric low-spin oxygenase domain (λ(max): 419,538, 568 nm) and incubation for 2-3 days at 4 °C converted the domain to a native enzyme-type, predominantly high-spin state (λ(max): ~395, ~512, ~650 nm). 7,8-Dihydrobiopterin and other thiols (e.g., β-mercaptoethanol, cysteine, and glutathione, with less effectiveness) can replace BH4 and DTT, respectively. The UV-visible absorption spectrum of L-Arg-bound ferric full-length nNOS, which exhibits a relatively intense band at ~650 nm (ε = 7.5-8 mM-1 cm-1) due to the presence of a neutral flavin semiquinone, can then be quantitatively reconstructed by combining the spectra of equimolar amounts of the oxygenase and reductase domains. Of particular note, the heme spin-state conversion does not occur in the absence of a thiol even after prolonged (35-48 h) incubation of the oxygenase domain with BH4 and/or L-Arg under anaerobic conditions. Thus, DTT (or other thiols) plays a significant role(s) beyond keeping BH4 in its reduced form, in restoring the pterin- and/or substrate- binding capability of the E. coli-expressed, BH4-free, dimeric nNOS oxygenase domain. Our results in combination with recently available X-ray crystallography and site-directed mutagenesis data suggest that the observed DTT effects arise from the involvement of an intersubunit disulfide bond or its rearrangement in the NOS dimer.
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UR - http://www.scopus.com/inward/citedby.url?scp=0033619706&partnerID=8YFLogxK
U2 - 10.1021/bi991580j
DO - 10.1021/bi991580j
M3 - Article
C2 - 10625450
AN - SCOPUS:0033619706
VL - 38
SP - 15853
EP - 15862
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 48
ER -