Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen

The heme domain (iNOS_heme) 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 iNOS_heme 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 iNOS_heme 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 iNOS_heme 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.