ChemInform Abstract: A MIXED‐METAL DERIVATIVE OF LACCASE CONTAINING MERCURY(II) IN THE TYPE 1 BINDING SITE

Morie-Bebel, M. Michelle; Morris, Margaret C.; Menzie, Janelle L.; McMillin, David R.

doi:10.1002/chin.198438258

Cited by 3 publications

(4 citation statements)

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“…Rhus vernicifera laccase (native Lc) was isolated from acetone powder (Saito and Co., Osaka, Japan) according to published procedures. , Laccase activity was assayed spectrophotometrically using N,N -dimethyl- p -phenylenediamine as a substrate . The T1Hg-substituted derivative (T1HgLc), in which the T1 Cu is removed and replaced by Hg 2+ , was prepared as described previously. − Both the native laccase and T1HgLc were characterized by absorption and EPR. UV/visible absorption spectra were recorded on a Hewlett-Packard HP8452A diode array spectrophotometer in either 1, 0.2, or 0.1 cm quartz cuvettes.…”

Section: Methodsmentioning

confidence: 99%

“…To focus specifically on the reaction of the T2/T3 trinuclear Cu cluster with O 2 , a derivative of the enzyme was prepared in which the T1 Cu was removed and replaced by a spectroscopically silent and redox inactive Hg 2+ . − This T1HgLc derivative provides two advantages: (1) it permits observation of the spectroscopic features of the trinuclear Cu center unobstructed by the T1 Cu, and (2) it deprives the enzyme of one of the electrons it needs to reduce O 2 to H 2 O. The fully (three-electron) reduced T1HgLc was shown to react with O 2 to produce a peroxide-level intermediate ( P ) in which peroxide bridges between the reduced T2 Cu and oxidized T3 Cu .…”

Section: Introductionmentioning

confidence: 99%

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Decay of the Peroxide Intermediate in Laccase: Reductive Cleavage of the O−O Bond

2001

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Laccase is a multicopper oxidase that contains four Cu ions, one type 1, one type 2, and a coupled binuclear type 3 Cu pair. The type 2 and type 3 centers form a trinuclear Cu cluster that is the active site for O(2) reduction to H(2)O. To examine the reaction between the type 2/type 3 trinuclear cluster and dioxygen, the type 1 Cu was removed and replaced with Hg(2+), producing the T1Hg derivative. When reduced T1Hg laccase is reacted with dioxygen, a peroxide intermediate (P) is formed. The present study examines the kinetics and mechanism of formation and decay of P in T1HgLc. The formation of P was found to be independent of pH and did not involve a kinetic solvent isotope effect, indicating that no proton is involved in the rate-determining step of formation of P. Alternatively, pH and isotope studies on the decay of P revealed that a proton enhances the rate of decay by 10-fold at low pH. This process shows an inverse k(H)/k(D) kinetic solvent isotope effect and involves protonation of a nearby residue that assists in catalysis, rather than direct protonation of the peroxide. Decay of P also involves a significant oxygen isotope effect (k(16)O(2)/k(18)O(2)) of 1.11 +/- 0.05, indicating that reductive cleavage of the O-O bond is the rate-determining step in the decay of P. The activation energy for this process was found to be approximately 9.0 kcal/mol. The exceptionally slow rate of decay of P is explained by the fact that this process involves a 1e(-) reductive cleavage of the O-O bond and there is a large Franck-Condon barrier associated with this process. Alternatively, the 2e(-) reductive cleavage of the O-O bond has a much larger driving force which minimizes this barrier and accelerates the rate of this reaction by approximately 10(7) in the native enzyme. This large difference in rate for the 2e(-) versus 1e(-) process supports a molecular mechanism for multicopper oxidases in which O(2) is reduced to H(2)O in two 2e(-) steps.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Decay of the Peroxide Intermediate in Laccase: Reductive Cleavage of the O−O Bond

2001

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“…The mercury derivative of lacease was prepared as before. 20 The Hg(II) derivatives of the other proteins were prepared by the addition of 1-2 equiv of Hg(II) to the apoprotein followed by dialysis to remove excess metal. In all cases the mercury to protein stoichiometry was 1.0 ± 0.1.…”

Section: Methodsmentioning

confidence: 99%

Structural characterization of mercury-substituted copper proteins. Results from x-ray absorption spectroscopy

et al. 1989

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“…In addition to the T2D derivative, a lacease derivative containing Hg2+ substituted into the type I site (TIHg) has been developed by McMillin and co-workers. 20 As prepared, TIHg contains an oxidized type 2 Cu, and the presence of a 330-nm absorption band20 suggests that the type 3 site is also oxidized. Fluoride binds to the type 2 Cu in T1 Hg in a manner similar to that observed in native lacease.…”

mentioning

confidence: 98%

Reactivity of the laccase trinuclear copper active site with dioxygen: an x-ray absorption edge study

Cole¹,

Tan²,

Yang³

et al. 1990

J. Am. Chem. Soc.

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ChemInform Abstract: A MIXED‐METAL DERIVATIVE OF LACCASE CONTAINING MERCURY(II) IN THE TYPE 1 BINDING SITE

Cited by 3 publications

References 1 publication

Decay of the Peroxide Intermediate in Laccase: Reductive Cleavage of the O−O Bond

Decay of the Peroxide Intermediate in Laccase: Reductive Cleavage of the O−O Bond

Structural characterization of mercury-substituted copper proteins. Results from x-ray absorption spectroscopy

Reactivity of the laccase trinuclear copper active site with dioxygen: an x-ray absorption edge study

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