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-, tetrahydrobio pterin (BH4)-, and flavin-containing monooxygenases which catalyze the NADP H-dependent conversion of L-arginine (L-Arg) to NO and citrulline. NOS cons ists of two domians: an N-terminal oxygenase (heme- and BH4-bound) domain a nd a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we ha ve spectroscopically examined the binding of L-Arg and BH4 to the dimeric, BH4-free ferric neuronal NOS (nNOS) oxygenase domain expressed in Escherich ia coli separately from the reductase domain. Addition of L-Arg or its anal ogue 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(l ambda(max): 419, 538, 568 nm) and incubation for 2-3 days at 4 degrees C co nverted the domain to a native enzyme-type, predominantly high-spin state(l ambda(max): similar to 395, similar to 512, similar to 650 nm), 7,8-Dihydro biopterin and other thiols (e.g., beta-mercaptoethanol, cysteine, and gluta thione, with less effectiveness) can replace BH4 and DTT, respectively. The UV-visible absorption spectrum of L-Arg-bound ferric full-length nNOS, whi ch exhibits a relatively intense band at similar to 650 nm (epsilon = 7.5-8 mM(-1) cm(-1)) due to the presence of a neutral flavin semiquinone, can th en be quantitatively reconstructed by combining the spectra of equimolar am ounts 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 p rolonged (35-48 h) incubation of the oxygenase domain with BH4 and/or L-Arg under anaerobic conditions. Thus, DTT (or other thiols) plays a significan t 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, dim eric nNOS oxygenase domain. Our results in combination with recently availa ble X-ray crystallography and site-directed mutagenesis data suggest that t he observed DTT effects arise from the involvement of an intersubunit disul fide bond or its rearrangement in the NOS dimer.