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.
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.
Sono
M
M
Department of Chemistry and Biochemistry and School of Medicine, University of South Carolina, Columbia, South Carolina 29208, USA. msono@psc.sc.edu
Ledbetter
A P
AP
McMillan
K
K
Roman
L J
LJ
Shea
T M
TM
Masters
B S
BS
Dawson
J H
JH
D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives*; D002942:Circular Dichroism; D004229:Dithiothreitol / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006418:Heme / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry; D052248:Nitric Oxide Synthase Type I; D010105:Oxygenases / Q000737:chemistry; D011485:Protein Binding; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014961:X-Ray Diffraction
D013438:Sulfhydryl Compounds; D001708:Biopterins; D006418:Heme; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D004229:Dithiothreitol