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A. R. Hurshman, C. Krebs, D. E. Edmondson, B. H. Huynh, and M. A. Marletta. 2000. Formation of a Pterin Radical in the Reaction of the Heme Domain of Inducible Nitric Oxide Synthase with Oxygen. Biochemistry 38: 15689-15696. Full Article

The heme domain (iNOSheme) of inducible nitric oxide synthase (NOS) was expressed in Escherichia coli and purified to homogeneity. Rapid freeze-quench (RFQ) EPR was used to monitor the reaction of the reduced iNOSheme with oxygen in the presence and absence of substrate. In these reactions, heme oxidation occurs at a rate of ~15 s-1 at 4 C. A transient species with a g = 2.0 EPR signal is also observed under these conditions. The spectral properties of the g = 2.0 signal are those of an anisotropic organic radical with S = 1/2. Comparison of the EPR spectra obtained when iNOSheme is reconstituted with N5-14N- and 15N-substituted tetrahydrobiopterin (H4B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H3B�) of the bound H4B. Substitution of D2O for H2O reveals the presence of hyperfine-coupled exchangeable protons in the H4B radical. This radical forms at a rate of 15-20 s-1, with a slower decay rate that varies (0.12-0.7 s-1) depending on the substrate. At 127 ms, H3B� accumulates to a maximum of 80% of the total iNOSheme concentration in the presence of arginine but only to ~2.8% in the presence of NHA. Double-mixing RFQ experiments, where NHA is added after the formation of H3B�, show that NHA does not react rapidly with H3B� and suggest that NHA instead prevents the formation of the H4B radical. These data constitute the first direct evidence for an NOS-bound H3B� and are most consistent with a role for H4B in electron transfer in the NOS reaction.

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