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 journalArticle

Abstract

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)
JournalBiochemistry
Volume38
Issue number48
StatePublished - Nov 30 1999

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Pterins
Nitric Oxide Synthase Type II
Heme
Nitric Oxide Synthase
Paramagnetic resonance
Electrons
Oxygen
Arginine
Protons
Nitrogen
Substrates
Escherichia coli
Substitution reactions
Oxidation
Experiments
sapropterin

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Medicine(all)

Cite this

Hurshman, Amy R. ; Krebs, Carsten ; Edmondson, Dale E. ; Huynh, Boi Hanh ; Marletta, Michael A. / Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen. In: Biochemistry. 1999 ; Vol. 38, No. 48.
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abstract = "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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Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen. / Hurshman, Amy R.; Krebs, Carsten; Edmondson, Dale E.; Huynh, Boi Hanh; Marletta, Michael A.

In: Biochemistry, Vol. 38, No. 48, 30.11.1999.

Research output: Contribution to journalArticle

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T1 - Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen

AU - Hurshman, Amy R.

AU - Krebs, Carsten

AU - Edmondson, Dale E.

AU - Huynh, Boi Hanh

AU - Marletta, Michael A.

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Y1 - 1999/11/30

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