REACTION OF NEURONAL NITRIC-OXIDE SYNTHASE WITH OXYGEN AT LOW-TEMPERATURE - EVIDENCE FOR REDUCTIVE ACTIVATION OF THE OXY-FERROUS COMPLEX BYTETRAHYDROBIOPTERIN

The reaction of reduced NO synthase (NOS) with molecular oxygen was st udied at -30 degrees C. In the absence of substrate, the complex forme d between ferrous NOS and O-2 was sufficiently long lived for a precis e spectroscopic characterization. This complex displayed similar spect ral characteristics as the oxyferrous complex of cytochrome P450 (lamb da(max) = 416.5 nm), It then decomposed to the ferric state. The oxida tion of the flavin components was much slower and could be observed on ly at temperatures higher than -20 degrees C. In the presence of subst rate (L-arginine), another, 12-nm blue-shifted, intermediate spectrum was formed. The breakdown of the latter species resulted in the produc tion of N-omega-hydroxy-L-arginine in a stoichiometry of maximally 52% per NOS heme. This product formation took place also in the absence o f the reductase domain of NOS, Both formation of the blueshifted inter mediate and of N-omega-hydroxy-L-arginine required the presence of tet rahydrobiopterin (BH4). We propose that the blue-shifted intermediate is the result of reductive activation of the oxygenated complex, and t he electron is provided by BH4. These observations suggest that the re duction of the oxyferroheme complex may be the main function of BH4 in NOS catalysis.