Reaction of neuronal nitric-oxide synthase with oxygen at low temperature. Evidence for reductive activation of the oxy-ferrous complex by tetrahydrobiopterin.
The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at -30 degreesC. In the absence of substrate, the complex formed between ferrous NOS and O2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spectral characteristics as the oxyferrous complex of cytochrome P450 (lambda max = 416.5 nm). It then decomposed to the ferric state. The oxidation of the flavin components was much slower and could be observed only at temperatures higher than -20 degreesC. In the presence of substrate (L-arginine), another, 12-nm blue-shifted, intermediate spectrum was formed. The breakdown of the latter species resulted in the production of Nomega-hydroxy-L-arginine in a stoichiometry of maximally 52% per NOS heme. This product formation took place also in the absence of the reductase domain of NOS. Both formation of the blue-shifted intermediate and of Nomega-hydroxy-L-arginine required the presence of tetrahydrobiopterin (BH4). We propose that the blue-shifted intermediate is the result of reductive activation of the oxygenated complex, and the electron is provided by BH4. These observations suggest that the reduction of the oxyferroheme complex may be the main function of BH4 in NOS catalysis.
Bec
N
N
Institut National de la Santé et de la Recherche Scientifique, U 128, Institut Fédératif de Recherche 24, 34293 Montpellier, France.
Gorren
A C
AC
Voelker
C
C
Mayer
B
B
Lange
R
R
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D003080:Cold Temperature; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity
D011994:Recombinant Proteins; D001708:Biopterins; D019001:Nitric Oxide Synthase; C003402:sapropterin; D010100:Oxygen