| Abstract
| - In nitric oxide synthase (NOS), (6R)-tetrahydrobiopterin (H4B) binds near the heme and canreduce a heme−dioxygen intermediate (FeIIO2) during Arg 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]. Aconserved Trp engages in aromatic stacking with H4B, and its mutation inhibits NO synthesis. To examinehow this W457 impacts H4B redox function, we performed single turnover reactions with the mouseinducible NOS oxygenase domain (iNOSoxy) mutants W457F and W457A. Ferrous mutants containingArg and H4B were mixed with O2-containing buffer, and then heme spectral transitions, H4B radicalformation, and Arg hydroxylation were followed versus time. A heme FeIIO2 intermediate was observedin W457A and W457F and had normal spectral characteristics. However, its disappearance rate (6.5 s-1in W457F and 3.0 s-1 in W457A) was slower than in wild-type (12.5 s-1). Rates of H4B radical formation(7.1 s-1 in W457F and 2.7 s-1 in W457A) matched their rates of FeIIO2 disappearance, but were slowerthan radical formation in wild-type (13 s-1). The extent of H4B radical formation in the mutants wassimilar to wild-type, but their radical decayed 2−4 times faster. These kinetic changes correlated withslower and less extensive Arg hydroxylation by the mutants (wild-type > W457F > W457A). We concludethat W457 ensures a correct tempo of electron transfer from H4B to heme FeIIO2, possibly by stabilizingthe H4B radical. Proper control of these parameters may help maximize Arg hydroxylation and minimizeuncoupled O2 activation at the heme.
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