Formation of a protonated trihydrohiopterin radical cation in the first reaction cycle of neuronal and endothelial nitric oxide synthase detected by electron paramagnetic: Resonance spectroscopy

Nitric oxide synthase (EC 1.14.13.39; NOS) converts L-arginine into NO and L-citrulline in a two-step reaction with NW-hydroxy-L-arginine (NOHLA) as a n intermediate. The active site iron in NOS has thiolate axial heme-iron li gation as found in the related monooxygenase cytochrome P450. In NOS, tetra hydrobiopterin (BH4) is an essential cofactor for both steps, but its funct ion is controversial. Previous optical studies of the reaction between redu ced NOS with O-2 at -30 degreesC suggested that BH4 may serve as an one-ele ctron donor in the first cycle, implying formation of a trihydrobiopterin r adical. We investigated the same reaction under identical conditions with e lectron paramagnetic resonance spectroscopy. With BH4-containing full-lengt h neuronal NOS we obtained an organic free radical (g-value 2.0042) in the presence of Arg, and a similar radical was observed with the endothelial NO S oxygenase domain in the presence of Arg and BH4. Without substrate the ra dical yield was greatly (10x) diminished. Without BH4, or with NOHLA instea d of Arg, no radical was observed. With 6-methyltetrahydropterin or 5-methy l-BH4 instead of BH4, radicals with somewhat different spec-tra were formed . On the basis of simulations we assign the signals to trihydropterin radic al cations protonated at N5. This is the first study that demonstrates the formation of a protonated trihydrobiopterin radical with the constitutive i soforms of NOS, and the first time the radical was obtained without exogeno us BH4. These results offer strong support for redox cycling of BH, in the first reaction cycle of NOS catalysis (BH4 <-> BH3.H+).