Reactivity of tetrahydrobiopterin bound to nitric-oxide synthase.
Levels of tetrahydrobiopterin (BH(4)) bound to nitric-oxide synthase (NOS) were examined during multiple turnovers of the enzyme in the presence of an NADPH-regenerating system. Our findings show that NOS-bound BH(4) does not remain in a static state but undergoes redox reactions. Under these experimental conditions, the redox state of BH(4) was determined by the balance between calcium/calmodulin (Ca(2+)/CaM)-dependent oxidation of BH(4) mediated by the uncoupled formation of superoxide/hydrogen peroxide on the one hand and by reductive regeneration of BH(4) on the other hand. BH(4) oxidation was appreciably increased in the presence of arginine. Levels of NOS-bound BH(4) were also examined under single turnover conditions in the absence of an NADPH-regenerating system and in the presence of added superoxide dismutase and catalase to suppress the accumulation of superoxide and hydrogen peroxide. BH(4) oxidation was again dependent on Ca(2+)/CaM. The insensitivity to superoxide dismutase and catalase suggested that the single turnover oxidation of BH(4) did not proceed through superoxide/peroxide, although the involvement of these oxidants could not be definitively excluded. The amount of BH(4) oxidized was highest in the presence of arginine, and this oxidation significantly exceeded that in the presence of N(G)-hydroxy-L-arginine. The findings that single turnover oxidation of BH(4) is stimulated by arginine in the presence of Ca(2+)/CaM and that BH(4) is regenerated are consistent with a role for the pterin as an electron donor in product formation; this role remains to be defined.
Witteveen
C F
CF
Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20892-4096, USA.
Giovanelli
J
J
Kaufman
S
S
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D051381:Rats
D001708:Biopterins; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin