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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.
Biochemistry. 1999 Nov 30; 38(48):15853-62.B

Abstract

Nitric oxide (NO) synthases (NOS) are thiolate-ligated heme-, tetrahydrobiopterin (BH(4))-, and flavin-containing monooxygenases which catalyze the NADPH-dependent conversion of L-arginine (L-Arg) to NO AND citrulline. NOS consists of two domains: an N-terminal oxygenase (heme- and BH(4)-bound) domain and a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we have spectroscopically examined the binding of L-Agr and BH(4) to the dimeric, BH(4)-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 BH(4), together with dithiothreitol (DTT), to the pterin-free ferric low-spin oxygenase domain (gamma(MAX): 419, 538, 568 NM) and incubation for 2-3 days at 4 degrees C converted the domain to a native enzyme-type, predominantly high-spin state (gamma(MAX): approximately 395, approximately 512, approximately 650 NM). 7,8-Dihydrobiopterin and other thiols (E.G., beta-mercaptoethanol, cysteine, and glutathione, with less effectiveness) can replace BH(4) and DTT, respectively. the UV-visible absorption spectrum of L-Arg-bound ferric full length NNOS, which exhibits a relatively intense band at approximately 650 NM (epsilon equals 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 BH(4) and/or L-Arg under anaerobic conditions. Thus, DTT (or other thiols) plays a significant role(s) beyond keeping BH(4) in its reduced form, In restoring the pterin- and/or substrate-binding capability of the E. coli-expressed, BH(4) 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.

Authors+Show Affiliations

Sono M
Department of Chemistry and Biochemistry and School of Medicine, University of South Carolina, Columbia, South Carolina 29208, USA. msono@psc.sc.edu
Ledbetter AP
No affiliation info available
McMillan K
No affiliation info available
Roman LJ
No affiliation info available
Shea TM
No affiliation info available
Masters BS
No affiliation info available
Dawson JH
No affiliation info available

MeSH

Binding SitesBiopterinCircular DichroismDithiothreitolEscherichia coliHemeNitric Oxide SynthaseNitric Oxide Synthase Type IOxygenasesProtein BindingSubstrate SpecificitySulfhydryl CompoundsX-Ray Diffraction

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10625450

Citation

Sono, M, et al. "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." Biochemistry, vol. 38, no. 48, 1999, pp. 15853-62.
Sono M, Ledbetter AP, McMillan K, et al. 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. Biochemistry. 1999;38(48):15853-62.
Sono, M., Ledbetter, A. P., McMillan, K., Roman, L. J., Shea, T. M., Masters, B. S., & Dawson, J. H. (1999). 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. Biochemistry, 38(48), 15853-62.
Sono M, et al. 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. Biochemistry. 1999 Nov 30;38(48):15853-62. PubMed PMID: 10625450.
* Article titles in AMA citation format should be in sentence-case
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,M, AU - Ledbetter,A P, AU - McMillan,K, AU - Roman,L J, AU - Shea,T M, AU - Masters,B S, AU - Dawson,J H, PY - 2000/1/8/pubmed PY - 2000/1/8/medline PY - 2000/1/8/entrez SP - 15853 EP - 62 JF - Biochemistry JO - Biochemistry VL - 38 IS - 48 N2 - Nitric oxide (NO) synthases (NOS) are thiolate-ligated heme-, tetrahydrobiopterin (BH(4))-, and flavin-containing monooxygenases which catalyze the NADPH-dependent conversion of L-arginine (L-Arg) to NO AND citrulline. NOS consists of two domains: an N-terminal oxygenase (heme- and BH(4)-bound) domain and a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we have spectroscopically examined the binding of L-Agr and BH(4) to the dimeric, BH(4)-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 BH(4), together with dithiothreitol (DTT), to the pterin-free ferric low-spin oxygenase domain (gamma(MAX): 419, 538, 568 NM) and incubation for 2-3 days at 4 degrees C converted the domain to a native enzyme-type, predominantly high-spin state (gamma(MAX): approximately 395, approximately 512, approximately 650 NM). 7,8-Dihydrobiopterin and other thiols (E.G., beta-mercaptoethanol, cysteine, and glutathione, with less effectiveness) can replace BH(4) and DTT, respectively. the UV-visible absorption spectrum of L-Arg-bound ferric full length NNOS, which exhibits a relatively intense band at approximately 650 NM (epsilon equals 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 BH(4) and/or L-Arg under anaerobic conditions. Thus, DTT (or other thiols) plays a significant role(s) beyond keeping BH(4) in its reduced form, In restoring the pterin- and/or substrate-binding capability of the E. coli-expressed, BH(4) 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/10625450/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_ L2 - https://doi.org/10.1021/bi991580j DB - PRIME DP - Unbound Medicine ER -