The free radical site in pea seedling copper amine oxidase probed by resonance Raman spectroscopy and generated by photolysis of caged substrate

Johnson, Steven A.; Bisby, Roger H.; Tavender, Susan M.; Parker, Anthony W.

doi:10.1016/0014-5793(96)00045-2

Cited by 8 publications

(12 citation statements)

References 28 publications

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“…All of the spectra are dominated by an intense feature at ∼1650 cm -1 , as has also been observed for the semiquinone produced by the addition of benzylamine to PSAO (36). Our earlier work on ECAO proved that the peaks at ∼1450 and 1650 have partial ν-(CdN) character from their shifts of -3 and -4 cm -1 , respectively, with 15 N-labeled substrate (21).…”

Section: Resultssupporting

confidence: 68%

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

et al. 1997

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Resonance Raman (RR) spectroscopy has proven to be an excellent technique for providing structural information about the 2,4, 5-trihydroxyphenylalaninequinone (TPQ) cofactor and for identifying the source of oxygen atoms during the posttranslational synthesis of the cofactor. Through specific labeling of the C2, C4, and C5 oxygens of TPQ in phenylethylamine oxidase (PEAO) from Arthrobacter globiformis, we have identified the C=O stretch of the C5 carbonyl at 1683 cm-1 (-27 in 18O) and the C=O stretch of the C2 carbonyl at 1575 cm-1 (-21 in 18O). These vibrational frequencies show that the C-O moiety at C5 has far greater double-bond character than at C2 or C4, thereby explaining the exclusive nucleophilic attack at the C5 position by substrates and substrate analogs. Bovine serum amine oxidase (BSAO) exhibits a similar nu(C=O) mode at 1678 cm-1 (-22 cm-1 in 18O). Aniline reacts with the TPQ cofactor of PEAO to form a new derivative (lambdamax at 450 nm) with properties similar to the proposed substrate-imine intermediate in the catalytic cycle. It retains the C2=O spectral features of the native enzyme and exhibits a new C5=N stretch at 1603 cm-1 (-29 in 15N). In contrast, methylamine reacts with both PEAO and BSAO under anaerobic conditions to form a different stable adduct (lambdamax at 385 nm) with properties closer to the proposed product-imine intermediate in the catalytic cycle. This species has a distinctive RR spectrum with a C=N stretch at 1617 cm-1 that corresponds to the atoms of the added methylamine (-58 cm-1 with CD3NH2, -19 cm-1 with CH315NH2). The lack of D2O dependence of nu(C=N) shows that this is a deprotonated imine, which would be more stable toward hydrolysis than the postulated protonated imine in the enzymatic reaction. However, the BSAO product imine (from methylamine) does undergo hydrolysis and conversion to semiquinone upon addition of cyanide. It is possible that the inactive form of the product imine is stabilized by deprotonation and flipping of the TPQ ring [Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J. P. (1997) Biochemistry 36, 11472-11478].

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Section: Resultssupporting

confidence: 68%

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

et al. 1997

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“…The majority of the peaks in the Raman resonance spectrum of both the oxidized and reduced states of underivatized Cu‐AO from pea seedlings present shifts of 1200–1700 cm −1 . Some of the peaks attributed specifically to the semiquinolamine radical are the 1647 cm −1 (assigned to a C‐C stretching mode) and the 1469 cm −1 ones (assigned to a C‐O or C‐N stretching mode) [25]. Comparison with the Raman resonance spectra of model compounds has confirmed that the yellow radical species is indeed a semiquinolamine.…”

Section: The Radical Speciesmentioning

confidence: 87%

“…The aromatic hydrazone derivatives of the TPQ cofactor show characteristic resonance‐enhanced Raman bands which have been used to probe its chemical structure by comparison with model compounds [5,23]. Raman resonance spectroscopy with excitation in the 450–500 nm absorption bands also proves suitable to explore and characterize the quinone and semiquinolamine states of non‐derivatized TPQ [24,25]. The majority of the peaks in the Raman resonance spectrum of both the oxidized and reduced states of underivatized Cu‐AO from pea seedlings present shifts of 1200–1700 cm −1 .…”

Section: The Radical Speciesmentioning

confidence: 99%

Cu^I‐semiquinone radical species in plant copper‐amine oxidases

et al. 1999

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The intermediate CuI-semiquinone radical species in the catalytic mechanism of copper-amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped-flow and temperature-jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.

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“…This rapidly disappears upon admission of oxygen with a second order constant [11] of 2.5 x 107dm 3 mol-ls-1. The resonance Raman spectrum of this intermediate has been measured using both 14Nand 15N-containing substrates [5,12]. Small changes in the frequencies of some modes indicate incorporation of the nitrogen atom from substrate into the intermediate and identify it as a semi-iminoquinone.…”

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confidence: 99%

Time‐Resolved Resonance RamanStudies of Radicals From4‐Aminoresorcinol as Modelsfor the Active Site RadicalIntermediate in CopperAmine Oxidases

et al. 1999

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Radicals formed by one-electron oxidation of 4-aminoresorcinol have been studied as models for the active site free radical intermediate which forms as a stable product during anaerobic incubation of amine oxidases with amine substrates. Pulse radiolysis shows that the radical undergoes ionisations with pKa's of 3.4 and 6.4. Although the two deprotonated forms are difficult to distinguish by absorption spectroscopy, they have clearly different resonance Raman spectra. Comparison with the resonance Raman spectrum of the enzyme intermediate shows it to be the singly deprotonated form of the radical.

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The free radical site in pea seedling copper amine oxidase probed by resonance Raman spectroscopy and generated by photolysis of caged substrate

Cited by 8 publications

References 28 publications

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

Cu^I‐semiquinone radical species in plant copper‐amine oxidases

Time‐Resolved Resonance RamanStudies of Radicals From4‐Aminoresorcinol as Modelsfor the Active Site RadicalIntermediate in CopperAmine Oxidases

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The free radical site in pea seedling copper amine oxidase probed by resonance Raman spectroscopy and generated by photolysis of caged substrate

Cited by 8 publications

References 28 publications

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

CuI‐semiquinone radical species in plant copper‐amine oxidases

Time‐Resolved Resonance RamanStudies of Radicals From4‐Aminoresorcinol as Modelsfor the Active Site RadicalIntermediate in CopperAmine Oxidases

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Cu^I‐semiquinone radical species in plant copper‐amine oxidases