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 (