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

Amy R. Hurshman, Carsten Krebs, Dale E. Edmondson, Michael A. Marletta

Research output: Contribution to journalArticle

49 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)13287-13303
Number of pages17
Issue number45
StatePublished - Nov 18 2003


All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this