Reactivity of the flavin semiquinone of nitric oxide synthase in the oxygenation of arginine to NG-hydroxyarginine, the first step of nitric oxide synthesis.
Nitric oxide synthase (NOS) is a heme protein that catalyzes the oxygenation of L-arginine in the presence of NADPH to form nitric oxide, L-citrulline and NADP+, and proceeds via two partial reactions: 1) L-Arginine --> NG-hydroxy-L-arginine 2) NG-Hydroxy-L-arginine --> L-citrulline + nitric oxide Calmodulin, FAD, FMN and tetrahydrobiopterin are required for both reactions. Reactions 1 and 2 require the input of 2 and 1 electron equivalents, respectively. Under normal multiple turnover conditions, these electrons are ultimately derived from NADPH. We previously reported that NOS contains an endogenous reductant that, in the absence of NADPH, can support the single-turnover oxygenation of L-arginine to NG-hydroxy-L-arginine and a relatively small amount of L-citrulline [Campos, K. L., Giovanelli, J., and Kaufman, S. (1995) J. Biol. Chem. 270, 1721-1728]. This reductant has now been identified as the stable flavin semiquinone free radical (FSQ). Its oxidation appears to be coupled to the formation of NG-hydroxy-L-arginine and L-citrulline. The rate of FSQ oxidation is two orders of magnitude slower than the flux of electrons from NADPH through NOS during normal turnover of the enzyme, indicating that FSQ is not the proximal electron donor for heme under these conditions.
Witteveen
C F
CF
Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20892, USA.
Giovanelli
J
J
Yim
M B
MB
Gachhui
R
R
Stuehr
D J
DJ
Kaufman
S
S
D001120:Arginine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism*; D002956:Citrulline / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*
D005182:Flavin-Adenine Dinucleotide; D002956:Citrulline; D009569:Nitric Oxide; C029276:flavin semiquinone; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D010100:Oxygen