| Abstract
| - How 6R-tetrahydrobiopterin (H4B) participates in Arg hydroxylation as catalyzed by the nitricoxide synthases (NOSs) is a topic of current interest. Previous work with the oxygenase domain of inducibleNOS (iNOSoxy) demonstrated that H4B radical formation is kinetically coupled to disappearance of aninitial heme−dioxy intermediate and to Arg hydroxylation in a single turnover reaction run at 10 °C[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]. Here we used 5-methyl-H4B to investigate how pterin structure influencesradical formation and associated catalytic steps. In the presence of Arg, the heme−dioxy intermediate in5-methyl-H4B-bound iNOSoxy reacted at a rate of 35 s-1, which is 3-fold faster than with H4B. This wascoupled to a faster rate of 5-methyl-H4B radical formation (40 vs 12.5 s-1) and to a faster and moreproductive Arg hydroxylation. The EPR spectrum of the enzyme-bound 5-methyl-H4B radical had differenthyperfine structure than the bound H4B radical and exhibited a 3-fold longer half-life after its formation.A crystal structure of 5-methyl-H4B-bound iNOSoxy revealed that there are minimal changes inconformation of the bound pterin or in its interactions with the protein as compared to H4B. Together, weconclude the following: (1) The rate of heme−dioxy reduction is linked to pterin radical formation andis sensitive to pterin structure. (2) Faster heme−dioxy reduction increases the efficiency of Arghydroxylation but still remains rate limiting for the reaction. (3) The 5-methyl group influences heme−dioxy reduction by altering the electronic properties of the pterin rather than changing protein structureor interactions. (4) Faster electron transfer from 5-methyl-H4B may be due to increased radical stabilityafforded by the N-5 methyl group.
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