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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