TY - JOUR
T1 - Prokaryotic Expression of the Heme- and Flavin-Binding Domains of Rat Neuronal Nitric Oxide Synthase as Distinct Polypeptides
T2 - Identification of the Heme-Binding Proximal Thiolate Ligand as Cysteine-415
AU - McMillan, Kirk
AU - Masters, Bettie Sue Siler
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1995
Y1 - 1995
N2 - The heme- and flavin-binding domains of constitutive rat neuronal nitric oxide synthase (NOS) were expressed in Escherichia coli as distinct polypeptides with properties characteristic of the intact enzyme. The amino-terminal heme-binding domain (residues 1-714) was expressed using the expression vector pCW. The denatured molecular mass of the expressed protein was 80 kDa, and the protein was shown to be immunoreactive to rabbit anti-NOS IgG. The NOS hemoprotein exhibited a ferrous-carbon monoxide difference spectrum with a wavelength maximum at 445 nm. Spectral perturbation with L-arginine and BH4 elicited a type I difference spectrum, confirming the presence of binding sites for these molecules within the N-terminal NOS polypeptide. Site-directed mutagenesis was applied to the putative axial heme ligand, cysteine-415, generating the histidine mutant, which confirmed the identity of the proximal ligand. NOS flavoproteins, with (Cl, residues 715-1429) and without (C2, residues 749-1429) an amino-terminal calmodulin-binding motif, were expressed using the vector pPROK-1. The Cl and C2 flavoproteins were immunoreactive to anti-NOS IgG and were sized at approximately 80 kDa. Both of the purified flavoproteins exhibited optical absorbance properties typical of a flavin prosthetic group, with wavelength maxima at 380 and 450 nm, and were competent in NADPH-dependent electron transfer to cytochrome c, with observed rates of ~2-4 μmol/min/mg. The bacterial expression of the NO synthase heme-binding oxygenase and flavoprotein oxidoreductase domains as isolated proteins with specific properties of the intact enzyme represents an important development in structure-function studies of this complex enzyme.
AB - The heme- and flavin-binding domains of constitutive rat neuronal nitric oxide synthase (NOS) were expressed in Escherichia coli as distinct polypeptides with properties characteristic of the intact enzyme. The amino-terminal heme-binding domain (residues 1-714) was expressed using the expression vector pCW. The denatured molecular mass of the expressed protein was 80 kDa, and the protein was shown to be immunoreactive to rabbit anti-NOS IgG. The NOS hemoprotein exhibited a ferrous-carbon monoxide difference spectrum with a wavelength maximum at 445 nm. Spectral perturbation with L-arginine and BH4 elicited a type I difference spectrum, confirming the presence of binding sites for these molecules within the N-terminal NOS polypeptide. Site-directed mutagenesis was applied to the putative axial heme ligand, cysteine-415, generating the histidine mutant, which confirmed the identity of the proximal ligand. NOS flavoproteins, with (Cl, residues 715-1429) and without (C2, residues 749-1429) an amino-terminal calmodulin-binding motif, were expressed using the vector pPROK-1. The Cl and C2 flavoproteins were immunoreactive to anti-NOS IgG and were sized at approximately 80 kDa. Both of the purified flavoproteins exhibited optical absorbance properties typical of a flavin prosthetic group, with wavelength maxima at 380 and 450 nm, and were competent in NADPH-dependent electron transfer to cytochrome c, with observed rates of ~2-4 μmol/min/mg. The bacterial expression of the NO synthase heme-binding oxygenase and flavoprotein oxidoreductase domains as isolated proteins with specific properties of the intact enzyme represents an important development in structure-function studies of this complex enzyme.
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U2 - 10.1021/bi00011a025
DO - 10.1021/bi00011a025
M3 - Article
C2 - 7534476
AN - SCOPUS:0028931726
VL - 34
SP - 3686
EP - 3693
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 11
ER -