A conserved tryptophan 457 modulates the kinetics and extent of N-hydroxy-L-arginine oxidation by inducible nitric-oxide synthase

Abstract

In the oxygenase domain of mouse inducible nitricoxide synthase (iNOSoxy), a conserved tryptophan residue, Trp-457, regulates the kinetics and extent of L-Arg oxidation to Nω-hydroxy-L-arginine (NOHA) by controlling electron transfer between bound (6R)-tetrahydrobiopterin (H4B) cofactor and the enzyme heine FeIIO2 intermediate (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819-12825). To investigate whether NOHA oxidation to citrulline and nitric oxide (NO) is regulated by a similar mechanism, we performed single turnover reactions with wild type iNOSoxy and mutants W457F and W457A. Ferrous proteins containing NOHA plus H4B or NOHA plus 7,8-dihydrobiopterin (H2B), were mixed with O2-containing buffer, and then heme spectral transitions and product formation were followed versus time. All three proteins formed a FeIIO2 intermediate with identical spectral characteristics. In wild type, H4B increased the disappearance rate of the FeIIO2 intermediate relative to H2B, and its disappearance was coupled to the formation of a FeIIINO immediate product prior to formation of ferric enzyme. In W457F and W457A, the disappearance rate of the FeIIO2 intermediate was slower than in wild type and took place without detectable build-up of the heme FeIIINO immediate product. Rates of FeIIO2 disappearance correlated with rates of citrulline formation in all three proteins, and reactions containing H4B formed 1.0, 0.54, and 0.38 citrulline/heme in wild type, W457F, and W457A iNOSoxy, respectively. Thus, Trp-457 modulates the kinetics of NOHA oxidation by iNOSoxy, and this is important for determining the extent of citrulline and NO formation. Our findings support a redox role for H4B during NOHA oxidation to NO by iNOSoxy.