Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain

Nitric oxide (NO), an intercellular messenger and an immuno-cytotoxic agent, is synthesized by the family of nitric oxide synthases (NOS), which are thiolate-ligated, heme-containing monooxygenases that convert L-Arg to L-citrulline and NO in a tetrahydrobiopterin (BH4)-dependent manner, using NADPH as the electron donor. The dioxygen complex of the ferrous enzyme has been proposed to be a key intermediate in the NOS catalytic cycle. In this study, we have generated a stable ferrous-O-2 complex of the oxygenase domain of rat neuronal NOS (nNOS) by bubbling O-2 through a solution of the dithionite-reduced enzyme at -30 degrees C in a cryogenic solvent containing 50% ethylene glycol. The most stable dioxygen complex is obtained using the oxygenase domain which has been preincubated for an extended length of time at 4 degrees C with BH4/dithiothreitol and N-G-methyl-L-arginine, a substrate analogue inhibitor. The O-2 complex of the nNOS oxygenase domain thus prepared exhibits UV-visible absorption (maxima at 419 and 553 nm, shoulder at similar to 585 nm) and magnetic circular dichroism spectra that are nearly identical to those of ferrous-O-2 cytochrome P450-CAM. Our;spectral data are noticeably blue-shifted from those seen at 10 degrees C for a short-lived transient species (lambda(max) = 427 nm) for the nNOS oxygenase domain using stopped-now rapid-scanning spectroscopy [Abu-Soud, Ii. M., Gachhui, R., Raushel, F. M., and Stuehr, D. J. (1997) J. Biol, Chem. 272, 17349], but somewhat similar to those of a relatively stable O-2 adduct of L-Arg-free full-length nNOS (lambda(max) = 415-416.5 nm) generated at -30 degrees C [Bec, N., Gorren, A. C. F., Voelder, C., Mayer, B., and Lange, R. (1998) J. Biol. Chem. 273, 13502]. Compared with ferrous-O-2 P450-CAM, however, the ferrous-O-2 adduct of the nNOS oxygenase domain is considerably more autoxidizable and the O-2-CO exchange reaction is noticeably slower. The generation of a stable ferrous-O-2 adduct of the nNOS oxygenase domain, as described herein, will facilitate further mechanistic and spectroscopic investigations of this important intermediate.