Reactivity, electrochemical, and spectroscopic studies of type 2 copper-depleted Rhus vernicifera laccase

R. Max Wynn, David B. Knaff, Robert A. Holwerda

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Abstract

Spectroelectrochemical, circular dichroism (CD), and kinetic measurements have been performed on native and type 2 copper-depleted (T2D) tree laccases to determine the effect of type 2 copper removal on the tertiary structure, blue copper reduction potential, and electron-transfer reactivity of the metalloenzyme. The midpoint potentials (E0) of type 1 Cu in T2Dr (reduced type 3 center) and native laccases, determined with (hydroxyethyl)ferrocene (HEF) as mediator, are essentially identical (430 and 429 mV, respectively; 25°C, pH 7.0, I = 0.5 M). Comparisons of the 300-700-nm CD spectra of native, T2Dr, and T2Dox (oxidized type 3 center) laccases reveal no detectable structural change at the type 1 copper center associated with removal of the type 2 Cu atom and only small effects of the oxidation state of the type 3 center on the environment of the type 1 site. Stopped-flow kinetic studies of electron transfer to T2Dr-type 1 Cu(II) from HEF and seven hydroquinones were performed to verify the importance of type 2 copper in the binding of polyphenolic substrates and to determine the effect of type 2 copper removal on the intrinsic electron-transfer reactivity of the blue copper atom. Removal of type 2 copper has remarkably little effect on the overall electron-transfer reactivity of substituted hydroquinones with laccase type 1 Cu(II), as measured by a second-order rate constant (k). The reduction rate of type 1 copper is slightly sensitive to the oxidation state of the type 3 center, as k values for electron transfer to T2Dr and T2Dox from hydroquinone are 5.2 × 102 and 3.2 × 102 M-1 s-1, respectively, at 25°C, pH 7.0, and I = 0.5 M. Detailed comparisons of rate-substrate concentration profiles show that ES complex formation constants are much smaller in T2Dr than in native laccase. The role of type 2 copper as a substrate binding site in the blue copper reduction pathway therefore is confirmed. The intrinsic electron-transfer reactivity of laccase type 1 copper increases substantially upon removal of the type 2 copper atom, as shown by rate constants for the HEF reduction of T2Dr-type 1 Cu(II) (1.9 × 104 M-1 s-1) and native type 1 Cu(II) (4.8 × 103 M-1 s-1), at 25°C, pH 7.0, and I = 0.5 M. Apparent blue copper self-exchange electron-transfer rate constants estimated from the application of relative Marcus theory to these HEF rate data are 1.6 and 2.5 × 101 M-1 s-1 for the native and T2D proteins, respectively.

Original languageEnglish (US)
Pages (from-to)241-247
Number of pages7
JournalBiochemistry
Volume23
Issue number2
StatePublished - 1984

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Rhus
Laccase
Copper
Electrons
Hydroquinones
Rate constants
Circular Dichroism
Atoms
Substrates
Oxidation
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reactivity, electrochemical, and spectroscopic studies of type 2 copper-depleted Rhus vernicifera laccase. / Wynn, R. Max; Knaff, David B.; Holwerda, Robert A.

In: Biochemistry, Vol. 23, No. 2, 1984, p. 241-247.

Research output: Contribution to journalArticle

Wynn, R. Max ; Knaff, David B. ; Holwerda, Robert A. / Reactivity, electrochemical, and spectroscopic studies of type 2 copper-depleted Rhus vernicifera laccase. In: Biochemistry. 1984 ; Vol. 23, No. 2. pp. 241-247.
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N2 - Spectroelectrochemical, circular dichroism (CD), and kinetic measurements have been performed on native and type 2 copper-depleted (T2D) tree laccases to determine the effect of type 2 copper removal on the tertiary structure, blue copper reduction potential, and electron-transfer reactivity of the metalloenzyme. The midpoint potentials (E0) of type 1 Cu in T2Dr (reduced type 3 center) and native laccases, determined with (hydroxyethyl)ferrocene (HEF) as mediator, are essentially identical (430 and 429 mV, respectively; 25°C, pH 7.0, I = 0.5 M). Comparisons of the 300-700-nm CD spectra of native, T2Dr, and T2Dox (oxidized type 3 center) laccases reveal no detectable structural change at the type 1 copper center associated with removal of the type 2 Cu atom and only small effects of the oxidation state of the type 3 center on the environment of the type 1 site. Stopped-flow kinetic studies of electron transfer to T2Dr-type 1 Cu(II) from HEF and seven hydroquinones were performed to verify the importance of type 2 copper in the binding of polyphenolic substrates and to determine the effect of type 2 copper removal on the intrinsic electron-transfer reactivity of the blue copper atom. Removal of type 2 copper has remarkably little effect on the overall electron-transfer reactivity of substituted hydroquinones with laccase type 1 Cu(II), as measured by a second-order rate constant (k). The reduction rate of type 1 copper is slightly sensitive to the oxidation state of the type 3 center, as k values for electron transfer to T2Dr and T2Dox from hydroquinone are 5.2 × 102 and 3.2 × 102 M-1 s-1, respectively, at 25°C, pH 7.0, and I = 0.5 M. Detailed comparisons of rate-substrate concentration profiles show that ES complex formation constants are much smaller in T2Dr than in native laccase. The role of type 2 copper as a substrate binding site in the blue copper reduction pathway therefore is confirmed. The intrinsic electron-transfer reactivity of laccase type 1 copper increases substantially upon removal of the type 2 copper atom, as shown by rate constants for the HEF reduction of T2Dr-type 1 Cu(II) (1.9 × 104 M-1 s-1) and native type 1 Cu(II) (4.8 × 103 M-1 s-1), at 25°C, pH 7.0, and I = 0.5 M. Apparent blue copper self-exchange electron-transfer rate constants estimated from the application of relative Marcus theory to these HEF rate data are 1.6 and 2.5 × 101 M-1 s-1 for the native and T2D proteins, respectively.

AB - Spectroelectrochemical, circular dichroism (CD), and kinetic measurements have been performed on native and type 2 copper-depleted (T2D) tree laccases to determine the effect of type 2 copper removal on the tertiary structure, blue copper reduction potential, and electron-transfer reactivity of the metalloenzyme. The midpoint potentials (E0) of type 1 Cu in T2Dr (reduced type 3 center) and native laccases, determined with (hydroxyethyl)ferrocene (HEF) as mediator, are essentially identical (430 and 429 mV, respectively; 25°C, pH 7.0, I = 0.5 M). Comparisons of the 300-700-nm CD spectra of native, T2Dr, and T2Dox (oxidized type 3 center) laccases reveal no detectable structural change at the type 1 copper center associated with removal of the type 2 Cu atom and only small effects of the oxidation state of the type 3 center on the environment of the type 1 site. Stopped-flow kinetic studies of electron transfer to T2Dr-type 1 Cu(II) from HEF and seven hydroquinones were performed to verify the importance of type 2 copper in the binding of polyphenolic substrates and to determine the effect of type 2 copper removal on the intrinsic electron-transfer reactivity of the blue copper atom. Removal of type 2 copper has remarkably little effect on the overall electron-transfer reactivity of substituted hydroquinones with laccase type 1 Cu(II), as measured by a second-order rate constant (k). The reduction rate of type 1 copper is slightly sensitive to the oxidation state of the type 3 center, as k values for electron transfer to T2Dr and T2Dox from hydroquinone are 5.2 × 102 and 3.2 × 102 M-1 s-1, respectively, at 25°C, pH 7.0, and I = 0.5 M. Detailed comparisons of rate-substrate concentration profiles show that ES complex formation constants are much smaller in T2Dr than in native laccase. The role of type 2 copper as a substrate binding site in the blue copper reduction pathway therefore is confirmed. The intrinsic electron-transfer reactivity of laccase type 1 copper increases substantially upon removal of the type 2 copper atom, as shown by rate constants for the HEF reduction of T2Dr-type 1 Cu(II) (1.9 × 104 M-1 s-1) and native type 1 Cu(II) (4.8 × 103 M-1 s-1), at 25°C, pH 7.0, and I = 0.5 M. Apparent blue copper self-exchange electron-transfer rate constants estimated from the application of relative Marcus theory to these HEF rate data are 1.6 and 2.5 × 101 M-1 s-1 for the native and T2D proteins, respectively.

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