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

Masanori Sono, Amy P. Ledbetter, Kirk McMillan, Linda J. Roman, Thomas M. Shea, Bettie Sue Siler Masters, John H. Dawson

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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 languageEnglish (US)
Pages (from-to)15853-15862
Number of pages10
JournalBiochemistry
Volume38
Issue number48
DOIs
StatePublished - Nov 30 1999

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Pterins
Oxygenases
Nitric Oxide Synthase Type I
Heme
Sulfhydryl Compounds
Escherichia coli
Dithiothreitol
Nitric Oxide Synthase
Substrates
Enzymes
dimethylaniline monooxygenase (N-oxide forming)
FMN Reductase
Oxidoreductases
Heme Oxygenase (Decyclizing)
Citrulline
Mutagenesis
Flavin-Adenine Dinucleotide
Mercaptoethanol
X ray crystallography
X Ray Crystallography

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 journalArticle

Sono, Masanori ; Ledbetter, Amy P. ; McMillan, Kirk ; Roman, Linda J. ; Shea, Thomas M. ; Masters, Bettie Sue Siler ; Dawson, John H. / 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. In: Biochemistry. 1999 ; Vol. 38, No. 48. pp. 15853-15862.
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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.",
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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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