Characterization of the topa quinone cofactor in amine oxidase from Escherichia coli by resonance Raman spectroscopy

Pierre Moenne-Loccoz, Nobuhumi Nakamura, Vincent Steinebach, Johannis A. Duine, Minae Mure, Judith P. Klinman, Joann Sanders-Loehr

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Abstract

The aromatic amine oxidase from Escherichia coli (ECAO) utilizes Cu(II) and 2,4,5-trihdroxyphenylalanine quinone (TPQ) as cofactors in enzymatic catalysis. The TPQ cofactor is clearly identified by a set of characteristic vibrational modes between 1200 and 1700 cm-1 in the resonance Raman (RR) spectrum of the native enzyme. This is the first report of a RR spectrum for an underivatized TPQ cofactor in an enzyme, showing that it is possible to study changes in the cofactor during the natural reaction cycle. The RR spectrum of ECAO closely matches that of a 2-hydroxy-1 ,4-benzoquinone model compound, particularly in the deprotonated state in aqueous solution. The principal in-phase C=O symmetric stretching mode of the quinone occurs at 1681 cm-1 in ECAO and at 1666 cm-1 in the model compound and, in both cases, undergoes a downshift of ∼25 cm-1 upon substitution of one of the carbonyl oxygens with 18O. The overall similarity of the 18O and D shifts in their RR spectra shows that the TPQ cofactor and model compound have the same structure and reactivity, with oxygen exchange occurring at the carbonyl adjacent to the hydroxyl group. Substrate reduction of ECAO under anaerobic conditions leads to a stable semiquinone (λmax at 442 and 468 nm) with a RR spectrum characteristic of an amine-substituted semiquinone. The intense ring mode at 1647 cm-1 undergoes a shift of -4 cm-1 in enzyme reacted with 15N-containing substrate and a shift of -6 cm-1 upon addition of cyanide, indicating that the semiquinone cofactor is perturbed by formation of the Cu(I)CN complex. Thus, the Cu(I) cofactor appears to be sufficiently close to undergo electrostatic interactions with the semiquinone cofactor in this catalytic intermediate of amine oxidase.

Original languageEnglish (US)
Pages (from-to)7020-7026
Number of pages7
JournalBiochemistry
Volume34
Issue number21
StatePublished - 1995

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Raman Spectrum Analysis
Escherichia coli
Amines
Raman spectroscopy
Raman scattering
Oxidoreductases
Enzymes
Oxygen
Coenzymes
Cyanides
Substrates
Coulomb interactions
Static Electricity
Catalysis
Hydroxyl Radical
Stretching
Substitution reactions
6-hydroxydopa quinone
benzoquinone

ASJC Scopus subject areas

  • Biochemistry

Cite this

Moenne-Loccoz, P., Nakamura, N., Steinebach, V., Duine, J. A., Mure, M., Klinman, J. P., & Sanders-Loehr, J. (1995). Characterization of the topa quinone cofactor in amine oxidase from Escherichia coli by resonance Raman spectroscopy. Biochemistry, 34(21), 7020-7026.

Characterization of the topa quinone cofactor in amine oxidase from Escherichia coli by resonance Raman spectroscopy. / Moenne-Loccoz, Pierre; Nakamura, Nobuhumi; Steinebach, Vincent; Duine, Johannis A.; Mure, Minae; Klinman, Judith P.; Sanders-Loehr, Joann.

In: Biochemistry, Vol. 34, No. 21, 1995, p. 7020-7026.

Research output: Contribution to journalArticle

Moenne-Loccoz, P, Nakamura, N, Steinebach, V, Duine, JA, Mure, M, Klinman, JP & Sanders-Loehr, J 1995, 'Characterization of the topa quinone cofactor in amine oxidase from Escherichia coli by resonance Raman spectroscopy', Biochemistry, vol. 34, no. 21, pp. 7020-7026.
Moenne-Loccoz, Pierre ; Nakamura, Nobuhumi ; Steinebach, Vincent ; Duine, Johannis A. ; Mure, Minae ; Klinman, Judith P. ; Sanders-Loehr, Joann. / Characterization of the topa quinone cofactor in amine oxidase from Escherichia coli by resonance Raman spectroscopy. In: Biochemistry. 1995 ; Vol. 34, No. 21. pp. 7020-7026.
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abstract = "The aromatic amine oxidase from Escherichia coli (ECAO) utilizes Cu(II) and 2,4,5-trihdroxyphenylalanine quinone (TPQ) as cofactors in enzymatic catalysis. The TPQ cofactor is clearly identified by a set of characteristic vibrational modes between 1200 and 1700 cm-1 in the resonance Raman (RR) spectrum of the native enzyme. This is the first report of a RR spectrum for an underivatized TPQ cofactor in an enzyme, showing that it is possible to study changes in the cofactor during the natural reaction cycle. The RR spectrum of ECAO closely matches that of a 2-hydroxy-1 ,4-benzoquinone model compound, particularly in the deprotonated state in aqueous solution. The principal in-phase C=O symmetric stretching mode of the quinone occurs at 1681 cm-1 in ECAO and at 1666 cm-1 in the model compound and, in both cases, undergoes a downshift of ∼25 cm-1 upon substitution of one of the carbonyl oxygens with 18O. The overall similarity of the 18O and D shifts in their RR spectra shows that the TPQ cofactor and model compound have the same structure and reactivity, with oxygen exchange occurring at the carbonyl adjacent to the hydroxyl group. Substrate reduction of ECAO under anaerobic conditions leads to a stable semiquinone (λmax at 442 and 468 nm) with a RR spectrum characteristic of an amine-substituted semiquinone. The intense ring mode at 1647 cm-1 undergoes a shift of -4 cm-1 in enzyme reacted with 15N-containing substrate and a shift of -6 cm-1 upon addition of cyanide, indicating that the semiquinone cofactor is perturbed by formation of the Cu(I)CN complex. Thus, the Cu(I) cofactor appears to be sufficiently close to undergo electrostatic interactions with the semiquinone cofactor in this catalytic intermediate of amine oxidase.",
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AB - The aromatic amine oxidase from Escherichia coli (ECAO) utilizes Cu(II) and 2,4,5-trihdroxyphenylalanine quinone (TPQ) as cofactors in enzymatic catalysis. The TPQ cofactor is clearly identified by a set of characteristic vibrational modes between 1200 and 1700 cm-1 in the resonance Raman (RR) spectrum of the native enzyme. This is the first report of a RR spectrum for an underivatized TPQ cofactor in an enzyme, showing that it is possible to study changes in the cofactor during the natural reaction cycle. The RR spectrum of ECAO closely matches that of a 2-hydroxy-1 ,4-benzoquinone model compound, particularly in the deprotonated state in aqueous solution. The principal in-phase C=O symmetric stretching mode of the quinone occurs at 1681 cm-1 in ECAO and at 1666 cm-1 in the model compound and, in both cases, undergoes a downshift of ∼25 cm-1 upon substitution of one of the carbonyl oxygens with 18O. The overall similarity of the 18O and D shifts in their RR spectra shows that the TPQ cofactor and model compound have the same structure and reactivity, with oxygen exchange occurring at the carbonyl adjacent to the hydroxyl group. Substrate reduction of ECAO under anaerobic conditions leads to a stable semiquinone (λmax at 442 and 468 nm) with a RR spectrum characteristic of an amine-substituted semiquinone. The intense ring mode at 1647 cm-1 undergoes a shift of -4 cm-1 in enzyme reacted with 15N-containing substrate and a shift of -6 cm-1 upon addition of cyanide, indicating that the semiquinone cofactor is perturbed by formation of the Cu(I)CN complex. Thus, the Cu(I) cofactor appears to be sufficiently close to undergo electrostatic interactions with the semiquinone cofactor in this catalytic intermediate of amine oxidase.

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