title: Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain.
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 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 NG-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 approximately 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 degrees C for a short-lived transient species (lambdamax = 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 (lambdamax = 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-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.
authors.affiliation: Department of Chemistry and Biochemistry, School of Medicine, University of South Carolina, Columbia 29208, USA.
authors.forename: A P
authors.initials: AP
authors.lastname: Ledbetter
authors.forename: K
authors.initials: K
authors.lastname: McMillan
authors.forename: L J
authors.initials: LJ
authors.lastname: Roman
authors.forename: B S
authors.initials: BS
authors.lastname: Masters
authors.forename: J H
authors.initials: JH
authors.lastname: Dawson
authors.forename: M
authors.initials: M
authors.lastname: Sono
mesh_terms: D019475:Camphor 5-Monooxygenase / Q000737:chemistry; D002248:Carbon Monoxide / Q000737:chemistry; D002942:Circular Dichroism; D003080:Cold Temperature; D005296:Ferrous Compounds / Q000737:chemistry*; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005296:Ferrous Compounds; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D002248:Carbon Monoxide; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D019475:Camphor 5-Monooxygenase; D010100:Oxygen