A conserved tryptophan 457 modulates the kinetics and extent of N-hydroxy-L-arginine oxidation by inducible nitric-oxide synthase.
In the oxygenase domain of mouse inducible nitric-oxide synthase (iNOSoxy), a conserved tryptophan residue, Trp-457, regulates the kinetics and extent of l-Arg oxidation to N(omega)-hydroxy-l-arginine (NOHA) by controlling electron transfer between bound (6R)-tetrahydrobiopterin (H(4)B) cofactor and the enzyme heme Fe(II)O(2) 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 H(4)B or NOHA plus 7,8-dihydrobiopterin (H(2)B), were mixed with O(2)-containing buffer, and then heme spectral transitions and product formation were followed versus time. All three proteins formed a Fe(II)O(2) intermediate with identical spectral characteristics. In wild type, H(4)B increased the disappearance rate of the Fe(II)O(2) intermediate relative to H(2)B, and its disappearance was coupled to the formation of a Fe(III)NO immediate product prior to formation of ferric enzyme. In W457F and W457A, the disappearance rate of the Fe(II)O(2) intermediate was slower than in wild type and took place without detectable build-up of the heme Fe(III)NO immediate product. Rates of Fe(II)O(2) disappearance correlated with rates of citrulline formation in all three proteins, and reactions containing H(4)B 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 H(4)B during NOHA oxidation to NO by iNOSoxy.
Wang
Zhi-Qiang
ZQ
Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA.
Wei
Chin-Chuan
CC
Stuehr
Dennis J
DJ
D002956:Citrulline / Q000138:chemical synthesis; D007700:Kinetics; D009569:Nitric Oxide / Q000138:chemical synthesis; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D014364:Tryptophan / Q000737:chemistry*
D002956:Citrulline; D009569:Nitric Oxide; D014364:Tryptophan; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II