Ability of Tetrahydrobiopterin Analogues to Support Catalysis by Inducible Nitric Oxide Synthase: Formation of a Pterin Radical is Required for Enzyme Activity

Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H₄B) or tetrahydrobiopterin analogues (5-methyl-H₄B and 4-amino-H₄B), and the ability of bound 5-methyl-H₄B and 4-amino-H₄B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiNOS) was examined. In a single turnover with HDiNOS, 5-methyl-H₄B forms a very stable radical, 5-methyl-H₃B′, that accumulates in the arginine reaction to ∼60% of the HDiNOS concentration and decays ∼400-fold more slowly than H₃B^. (0.0003 vs 0.12 s ^-1). The amount of radical (5-methyl-H₃B^. or H₃B^.) observed in the NHA reaction is very small (<3% of HDiNOS). The activity of 5-methyl-H₄B-saturated FLiNOS and HDiNOS is similar to that when H₄B is bound: arginine is hydroxylated to NHA, and NHA is oxidized exclusively to citrulline and ^.NO. A pterin radical was not observed with 4-amino-H₄B- or pterin-free HDiNOS with either substrate. The catalytic activity of 4-amino-H₄B-bound FLiNOS and HDiNOS resembles that of pterin-free iNOS: the hydroxylation of arginine is very unfavorable (<2% that of H₄B-bound iNOS), and NHA is oxidized to a mixture of amino acid products (citrulline and cyanoomithine) and NO^- rather than ^.NO. These results demonstrate that the bound pterin cofactor undergoes a one-electron oxidation (to form a pterin radical), which is essential to its ability to support normal NOS turnover. Although binding of H₄B also stabilizes the NOS structure and active site, the most critical role of the pterin cofactor in NOS appears to be in electron transfer.