Kinetic Studies of the Reduction of Rhus vernicifera Laccase

Author: Holwerda, Robert Alan

Year: 1974

Degree: Dissertation (Ph.D.)

Advisor: Gray, Harry B.

Committee Member: Unknown, Unknown

Option: Chemistry

DOI: 10.7907/k0xx-5286

Abstract

Anaerobic stopped flow kinetic studies of the reactions of hydroquinone, Fe(CN)64-, and Fe(EDTA)2- with Rhus vernicifera laccase have been performed with the objective of elucidating the mechanisms by which the three enzymatic copper sites are reduced.

Second order rate constants characterizing the hydroquinone reduction of laccase Type 1 and Type 3 copper sites at 25.6°, pH 7.0, μ = 0.1 (phosphate) are 325 and 457 M-1 sec-1, respectively. All of the experimental evidence points to the involvement of a common intermediate in the reduction mechanisms of these two functional units, and pH dependence results indicate that the phenolate monoanion HQ- is the active reductant interacting with the metalloprotein in the shared slow step. A requirement for penetration of HQ- into the first coordination sphere of Type 2 Cu(II) is strongly suggested by pH 6.0 fluoride inhibition results, and activation parameters (Type 1: ΔH‡ = 15.0 kcal/mole, ΔS‡ = +13 cal/mol-deg; Type 3: ΔH‡ = 13.6 kcal/mole, ΔS‡ = +15 cal/mol-deg; pH 7.0, μ = 0.1) are compatible with rate-limiting reduction of an essentially axial copper(II) species.

Decolorization of the intense peak with maximum at 405 nm characteristic of the Type 2 Cu(II)-N3- laccase derivative is first order in [N3-] , zeroth order in [hydroquinone], suggesting that azide bridging is involved in an intramolecular Type 1 Cu(I) to Type 2 Cu(II) electron transfer step. Attack at the Type 2 copper is not a prerequisite to reduction of the Type 1 site in the laccase-azide complex, but the reactivity of the "blue" copper is considerably altered from that in the native enzyme.

Ferrocyanide reduces laccase by a mechanism much different than that preferred by hydroquinone. The reduction rates of the Type 1 and Type 3 sites are identical (k = 42 M-1 sec-1, 25°, pH 7.0, μ = 0.1; ΔH‡ = 18.4 kcal/mole, ΔS‡ = +10 cal/mol-deg), and only three electron equivalents are transferred to the metalloprotein anaerobically. The Type 2 copper atom is not reduced by Fe(CN)64-. Ionic strength and pH dependence studies suggest that electrostatic factors direct Fe(CN)64- to its initial attack site. At least one histidine residue appears to be involved in outer-sphere complex formation between laccase and the highly anionic reducing agent. The rate-limiting step for ferrocyanide reduction of laccase is reversible; calculations based on the forward and reverse rate constants indicate that the "blue" copper is probably the initial attack site preferred by ferrocyanide. The unusually high activation enthalpy for the reduction of laccase "blue" copper by Fe(CN)64- is attributed to the inaccesibility of this site to direct attack by external reducing agents.

Preliminary experiments with Fe(EDTA)2- as reductant have shown that the laccase "blue" copper site is reduced over 1000 times slower than that of stellacyanin (k = 2.31 x 102 M-1 sec-1 (laccase), 4.2 x 105 M-1 sec-1 (stellacyanin); 25.1°, pH 6.0, μ = 0.5). Activation parameters for the reduction of laccase Type 1 copper by Fe(EDTA)2- are: ΔH‡ =13.0 kcal/mole, ΔS‡ = -4 cal/mol-deg; pH 7.0, μ = 0.1.

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