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

Amy P. Ledbetter, Kirk McMillan, Linda J. Roman, Bettie Sue Siler Masters, John H. Dawson, Masanori Sono

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)8014-8021
Number of pages8
JournalBiochemistry
Volume38
Issue number25
DOIs
StatePublished - Jun 22 1999

ASJC Scopus subject areas

  • Biochemistry

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