Abstract
Rat cerebellar nitric oxide synthase (NOS) purified from transfected human kidney cells catalyzes an NADPHdependent reduction of quinonoid dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4). Reduction of qBH2 at 25 microM 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 qBH2. 7,8-Dihydrobiopterin had negligible activity. Neither 7,8-dihydrobiopterin nor BH4 affected the rate of qBH2 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 qBH2 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 qBH2 to the essential role of BH4 in NOS is discussed.
Highlights
Part of the protein contains the heme, BH4, and argininebinding sites (4 – 8)
In the aromatic amino acid hydroxylases, BH4 acts as a cofactor, providing electrons for oxygenation; the oxidized biopterin, quinonoid dihydrobiopterin, is recycled to BH4 by dihydropteridine reductase [10]
Stimulation by qBH2 and BH4 of nitric oxide synthase (NOS) Activity and NG-Hydroxyarginine Synthesis—NOS activity was measured as described previously [15] with the exceptions that the reaction mixture was buffered with 25 mM sodium bicine, pH 7.6, NADPH concentration was decreased to 1 M in the presence of the NADPH regenerating system described above, pterin was added at the concentrations specified, and incubation was for 10 min at 37 °C
Summary
Vol 271, No 8, Issue of February 23, pp. 4143–4147, 1996 Printed in U.S.A. Reduction of Quinonoid Dihydrobiopterin to Tetrahydrobiopterin by Nitric Oxide Synthase*. On the basis of these results, it was suggested that BH4 may function as an allosteric effector or to maintain some group(s) on the enzyme in a reduced state required for activity [15] This suggestion has been supported by more recent studies showing that BH4 helps stabilize the active dimeric state of the enzyme [17, 18], maintains a stable heme pocket that is accessible to oxygen [19], and prevents and reverses the inhibition of NOS by nitric oxide [20]. Because it is not clear whether BH4 undergoes redox reactions during NOS catalysis, we have examined directly the reduction by NOS of qBH2 to BH4
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