A conserved tryptophan in nitric oxide synthase regulates heme-dioxy reduction by tetrahydrobiopterin

In nitric oxide synthase (NOS), (6R)-tetrahydrobiopterin (H4B) binds near t he heme and can reduce a heme-dioxygen intermediate ((FeO2)-O-II) during Ar g hydroxylation [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C ., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276. 315-319]. A cons erved Trp engages in aromatic stacking with H4B, and its mutation inhibits NO synthesis. To examine how this W457 impacts H4B redox function, we perfo rmed single turnover reactions with the mouse inducible NOS oxygenase domai n (iNOSoxy) mutants W457F and W457A. Ferrous mutants containing Arg and H4B were mixed with O-2-containing buffer, and then heme spectral transitions, H4B radical formation, and Arg hydroxylation were followed versus time. A heme (FeO2)-O-II intermediate was observed in W457A and W457F and had norma l spectral characteristics. However, its disappearance rate (6.5 s(-1) in W 457F and 3.0 s(-1) in W457A) was slower than in wild-type (12.5 s(-1)). Rat es of H4B radical formation (7.1 s(-1) in W457F and 2.7 s(-1) in W457A) mat ched their rates of (FeO2)-O-II disappearance, but were slower than radical formation in wild-type (13 s(-1)). The extent of H4B radical formation in the mutants was similar to wild-type, but their radical decayed 2-4 times f aster. These kinetic changes correlated with slower and less extensive Arg hydroxylation by the mutants (wild-type > W457F > W457A). We conclude that W457 ensures a correct tempo of electron transfer from H4B to heme (FeO2)-O -II, possibly by stabilizing the H4B radical. Proper control of these param eters may help maximize Arg hydroxylation and minimize uncoupled O-2 activa tion at the heme.