Reduction of quinonoid dihydrobiopterin to tetrahydrobiopterin by nitric oxide synthase

Rat cerebellar nitric oxide synthase (NOS) purified from transfected human kidney cells catalyzes an NADPH-dependent reduction of quinonoid dihydrobiopterin (qBH(2)) to tetrahydrobiopterin (BH4). Reduction of qBH(2) at 25 mu M proceeds at a rate that is comparable with that of the overall reaction (citrulline synthesis) and requires calcium ions and calmodulin for optimal activity; NADH has only 10% of the activity of NADPH. The reduction rate with the quinonoid form of 6-methyldihydropterin is approximately twice that with qBH(2). 7,8-Dihydrobiopterin had negligible activity. Neither 7,8-dihydrobiopterin nor BH4 affected the rate of qBH(2) reduction. Reduction is inhibited by the flavoprotein inhibitor diphenyleneiodonium, whereas inhibitors of electron transfer through heme (7-nitroindazole and N-nitroarginine) stimulated the rate to a small extent, Methotrexate, which inhibits a variety of enzymes catalyzing dihydrobiopterin reduction, did not inhibit. These studies provide the first demonstration of the reduction of qBH(2) to BH4 by NOS and indicate that the reduction is catalyzed by the flavoprotein ''diaphorase'' activity of NOS. This activity is located on the reductase (C-terminal) domain, whereas the high affinity BH4 site involved in NOS activation is located on the oxygenase (N-terminal) domain. The possible significance of this reduction of qBH(2) to the essential role of BH4 in NOS is discussed.