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

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

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Issue number48
StatePublished - Nov 30 1999

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • General Medicine


Dive into the research topics of 'Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen'. Together they form a unique fingerprint.

Cite this