TY - JOUR
T1 - Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain
AU - Ledbetter, Amy P.
AU - McMillan, Kirk
AU - Roman, Linda J.
AU - Masters, Bettie Sue Siler
AU - Dawson, John H.
AU - Sono, Masanori
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1999/6/22
Y1 - 1999/6/22
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0033594933&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033594933&partnerID=8YFLogxK
U2 - 10.1021/bi990619h
DO - 10.1021/bi990619h
M3 - Article
C2 - 10387045
AN - SCOPUS:0033594933
VL - 38
SP - 8014
EP - 8021
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 25
ER -