Fourier transform infrared characterization of a CuB-nitrosyl complex in cytochrome ba3 from Thermus thermophilus: Relevance to NO reductase activity in heme-copper terminal oxidases

Takahiro Hayashi, I. Jin Lin, Ying Chen, James A. Fee, Pierre Moenne-Loccoz

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Abstract

The two heme-copper terminal oxidases of Thermus thermophilus have been shown to catalyze the two-electron reduction of nitric oxide (NO) to nitrous oxide (N2O) [Giuffre, A.; Stubauer, G.; Sarti, P.; Brunori, M.; Zumft, W. G.; Buse, G.; Soulimane, T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14718-14723]. While it is well-established that NO binds to the reduced heme a3 to form a low-spin heme {FeNO}7 species, the role CuB plays in the binding of the second NO remains unclear. Here we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to fully reduced cytochrome ba3. Low-temperature UV-vis, EPR, and RR spectroscopies confirm the binding of NO to the heme a3 and the efficiency of the photolysis at 30 K. The ν(NO) modes from the light-induced FTIR difference spectra are isolated from other perturbed vibrations using 15NO and 15N 18O. The ν(N-O)a3 is observed at 1622 cm-1, and upon photolysis, it is replaced by a new ν(N-O) at 1589 cm-1 assigned to a CuB-nitrosyl complex. This N-O stretching frequency is more than 100 cm-1 lower than those reported for Cu-NO models with three N-ligands and for CuB +-NO in bovine aa3. Because the UV-vis and RR data do not support a bridging configuration between CuB and heme a3 for the photolyzed NO, we assign the exceptionally low ν(NO) to an O-bound (η1-O) or a side-on (η2-NO) CuB-nitrosyl complex. From this study, we propose that, after binding of a first NO molecule to the heme a3 of fully reduced Tt ba3, the formation of an N-bound {CuNO}11 is prevented, and the addition of a second NO produces an O-bond Cu B-hyponitrite species bridging CuB and Fea3. In contrast, bovine cytochrome c oxidase is believed to form an N-bound Cu B-NO species; the [{FeNO}7{CuNO}11] complex is suggested here to be an inhibitory complex.

Original languageEnglish (US)
Pages (from-to)14952-14958
Number of pages7
JournalJournal of the American Chemical Society
Volume129
Issue number48
DOIs
StatePublished - Dec 5 2007
Externally publishedYes

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Thermus thermophilus
Nitric oxide
Fourier Analysis
Heme
Copper
Fourier transforms
Oxidoreductases
Nitric Oxide
Infrared radiation
Proteins
Photolysis
nitric-oxide reductase
cytochrome ba3
copper oxidase
Fourier Transform Infrared Spectroscopy
Temperature
Nitrous Oxide
Electron Transport Complex IV
Vibration

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Fourier transform infrared characterization of a CuB-nitrosyl complex in cytochrome ba3 from Thermus thermophilus : Relevance to NO reductase activity in heme-copper terminal oxidases. / Hayashi, Takahiro; Lin, I. Jin; Chen, Ying; Fee, James A.; Moenne-Loccoz, Pierre.

In: Journal of the American Chemical Society, Vol. 129, No. 48, 05.12.2007, p. 14952-14958.

Research output: Contribution to journalArticle

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abstract = "The two heme-copper terminal oxidases of Thermus thermophilus have been shown to catalyze the two-electron reduction of nitric oxide (NO) to nitrous oxide (N2O) [Giuffre, A.; Stubauer, G.; Sarti, P.; Brunori, M.; Zumft, W. G.; Buse, G.; Soulimane, T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14718-14723]. While it is well-established that NO binds to the reduced heme a3 to form a low-spin heme {FeNO}7 species, the role CuB plays in the binding of the second NO remains unclear. Here we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to fully reduced cytochrome ba3. Low-temperature UV-vis, EPR, and RR spectroscopies confirm the binding of NO to the heme a3 and the efficiency of the photolysis at 30 K. The ν(NO) modes from the light-induced FTIR difference spectra are isolated from other perturbed vibrations using 15NO and 15N 18O. The ν(N-O)a3 is observed at 1622 cm-1, and upon photolysis, it is replaced by a new ν(N-O) at 1589 cm-1 assigned to a CuB-nitrosyl complex. This N-O stretching frequency is more than 100 cm-1 lower than those reported for Cu-NO models with three N-ligands and for CuB +-NO in bovine aa3. Because the UV-vis and RR data do not support a bridging configuration between CuB and heme a3 for the photolyzed NO, we assign the exceptionally low ν(NO) to an O-bound (η1-O) or a side-on (η2-NO) CuB-nitrosyl complex. From this study, we propose that, after binding of a first NO molecule to the heme a3 of fully reduced Tt ba3, the formation of an N-bound {CuNO}11 is prevented, and the addition of a second NO produces an O-bond Cu B-hyponitrite species bridging CuB and Fea3. In contrast, bovine cytochrome c oxidase is believed to form an N-bound Cu B-NO species; the [{FeNO}7{CuNO}11] complex is suggested here to be an inhibitory complex.",
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T1 - Fourier transform infrared characterization of a CuB-nitrosyl complex in cytochrome ba3 from Thermus thermophilus

T2 - Relevance to NO reductase activity in heme-copper terminal oxidases

AU - Hayashi, Takahiro

AU - Lin, I. Jin

AU - Chen, Ying

AU - Fee, James A.

AU - Moenne-Loccoz, Pierre

PY - 2007/12/5

Y1 - 2007/12/5

N2 - The two heme-copper terminal oxidases of Thermus thermophilus have been shown to catalyze the two-electron reduction of nitric oxide (NO) to nitrous oxide (N2O) [Giuffre, A.; Stubauer, G.; Sarti, P.; Brunori, M.; Zumft, W. G.; Buse, G.; Soulimane, T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14718-14723]. While it is well-established that NO binds to the reduced heme a3 to form a low-spin heme {FeNO}7 species, the role CuB plays in the binding of the second NO remains unclear. Here we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to fully reduced cytochrome ba3. Low-temperature UV-vis, EPR, and RR spectroscopies confirm the binding of NO to the heme a3 and the efficiency of the photolysis at 30 K. The ν(NO) modes from the light-induced FTIR difference spectra are isolated from other perturbed vibrations using 15NO and 15N 18O. The ν(N-O)a3 is observed at 1622 cm-1, and upon photolysis, it is replaced by a new ν(N-O) at 1589 cm-1 assigned to a CuB-nitrosyl complex. This N-O stretching frequency is more than 100 cm-1 lower than those reported for Cu-NO models with three N-ligands and for CuB +-NO in bovine aa3. Because the UV-vis and RR data do not support a bridging configuration between CuB and heme a3 for the photolyzed NO, we assign the exceptionally low ν(NO) to an O-bound (η1-O) or a side-on (η2-NO) CuB-nitrosyl complex. From this study, we propose that, after binding of a first NO molecule to the heme a3 of fully reduced Tt ba3, the formation of an N-bound {CuNO}11 is prevented, and the addition of a second NO produces an O-bond Cu B-hyponitrite species bridging CuB and Fea3. In contrast, bovine cytochrome c oxidase is believed to form an N-bound Cu B-NO species; the [{FeNO}7{CuNO}11] complex is suggested here to be an inhibitory complex.

AB - The two heme-copper terminal oxidases of Thermus thermophilus have been shown to catalyze the two-electron reduction of nitric oxide (NO) to nitrous oxide (N2O) [Giuffre, A.; Stubauer, G.; Sarti, P.; Brunori, M.; Zumft, W. G.; Buse, G.; Soulimane, T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14718-14723]. While it is well-established that NO binds to the reduced heme a3 to form a low-spin heme {FeNO}7 species, the role CuB plays in the binding of the second NO remains unclear. Here we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to fully reduced cytochrome ba3. Low-temperature UV-vis, EPR, and RR spectroscopies confirm the binding of NO to the heme a3 and the efficiency of the photolysis at 30 K. The ν(NO) modes from the light-induced FTIR difference spectra are isolated from other perturbed vibrations using 15NO and 15N 18O. The ν(N-O)a3 is observed at 1622 cm-1, and upon photolysis, it is replaced by a new ν(N-O) at 1589 cm-1 assigned to a CuB-nitrosyl complex. This N-O stretching frequency is more than 100 cm-1 lower than those reported for Cu-NO models with three N-ligands and for CuB +-NO in bovine aa3. Because the UV-vis and RR data do not support a bridging configuration between CuB and heme a3 for the photolyzed NO, we assign the exceptionally low ν(NO) to an O-bound (η1-O) or a side-on (η2-NO) CuB-nitrosyl complex. From this study, we propose that, after binding of a first NO molecule to the heme a3 of fully reduced Tt ba3, the formation of an N-bound {CuNO}11 is prevented, and the addition of a second NO produces an O-bond Cu B-hyponitrite species bridging CuB and Fea3. In contrast, bovine cytochrome c oxidase is believed to form an N-bound Cu B-NO species; the [{FeNO}7{CuNO}11] complex is suggested here to be an inhibitory complex.

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