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

204 Citations (Scopus)

Abstract

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- 14 N- and 15 N-substituted tetrahydrobiopterin (H 4 B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H 3 B·) of the bound H 4 B. Substitution of D 2 O for H 2 O reveals the presence of hyperfine- coupled exchangeable protons in the H 4 B 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, H 3 B· 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 H 3 B·, show that NHA does not react rapidly with H 3 B· and suggest that NHA instead prevents the formation of the H 4 B radical. These data constitute the first direct evidence for an NOS-bound H 3 B· and are most consistent with a role for H 4 B in electron transfer in the NOS reaction.

Original languageEnglish (US)
Pages (from-to)15689-15696
Number of pages8
JournalBiochemistry
Volume38
Issue number48
DOIs
StatePublished - Nov 30 1999

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

All Science Journal Classification (ASJC) codes

  • Biochemistry

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. pp. 15689-15696.
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abstract = "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- 14 N- and 15 N-substituted tetrahydrobiopterin (H 4 B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H 3 B·) of the bound H 4 B. Substitution of D 2 O for H 2 O reveals the presence of hyperfine- coupled exchangeable protons in the H 4 B 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, H 3 B· 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 H 3 B·, show that NHA does not react rapidly with H 3 B· and suggest that NHA instead prevents the formation of the H 4 B radical. These data constitute the first direct evidence for an NOS-bound H 3 B· and are most consistent with a role for H 4 B 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, p. 15689-15696.

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.

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AB - 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- 14 N- and 15 N-substituted tetrahydrobiopterin (H 4 B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H 3 B·) of the bound H 4 B. Substitution of D 2 O for H 2 O reveals the presence of hyperfine- coupled exchangeable protons in the H 4 B 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, H 3 B· 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 H 3 B·, show that NHA does not react rapidly with H 3 B· and suggest that NHA instead prevents the formation of the H 4 B radical. These data constitute the first direct evidence for an NOS-bound H 3 B· and are most consistent with a role for H 4 B in electron transfer in the NOS reaction.

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