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-O₂ complex of the oxygenase domain of rat neuronal NOS (nNOS) by bubbling O₂ through a solution of the dithionite-reduced enzyme at -30 °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 °C with BH₄/dithiothreitol and N(G)-methyl-L-arginine, a substrate analogue inhibitor. The O₂ complex of the nNOS oxygenase domain thus prepared exhibits UV-visible absorption (maxima at 419 and 553 nm, shoulder at ~585 nm) and magnetic circular dichroism spectra that are nearly identical to those of ferrous-O₂ cytochrome P450-CAM. Our spectral data are noticeably blue-shifted from those seen at 10 °C for a short-lived transient species (λ(max) = 427 nm) for the nNOS oxygenase domain using stopped-flow rapid-scanning spectroscopy [Abu-Soud, H. 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 O₂ adduct of L-Arg-free full-length nNOS (λ(max) = 415-416.5 nm) generated at -30 °C [Bec, N., Gorren, A. C. F., Voelder, C., Mayer, B., and Lange, R. (1998) J. Biol. Chem. 273, 13502]. Compared with ferrous-O₂ P450-CAM, however, the ferrous-O₂ adduct of the nNOS oxygenase domain is considerably more autoxidizable and the O₂-CO exchange reaction is noticeably slower. The generation of a stable ferrous- O₂ adduct of the nNOS oxygenase domain, as described herein, will facilitate further mechanistic and spectroscopic investigations of this important intermediate.