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
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DOI：
10.1021/ja010365z
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发表时间：
2001-07-11
影响因子：
15
通讯作者：
Solomon, EI
中科院分区：
文献类型：
--
作者：
Palmer, AE;Lee, SK;Solomon, EI
漆酶是一种多铜氧化酶，含有四个铜离子，一个1型，一个2型，和一个耦合的双核3型铜对。2型和3型中心形成三核Cu簇，其是O-2还原成H2O的活性位点。为了研究2型/3型三核簇与双氧之间的反应，将1型Cu去除并用Hg 2+取代，产生T1 Hg衍生物。当还原型T1 Hg漆酶与分子氧反应时，形成过氧化物中间体（P）。本研究探讨的动力学和机制的形成和衰减的P在T1汞Lc。发现P的形成不依赖于pH，并且不涉及动力学溶剂同位素效应，表明没有质子参与P形成的速率决定步骤。对P衰变的pH和同位素研究表明，在低pH下，质子使衰变速率提高10倍。动力学溶剂同位素效应，涉及附近残基的质子化，其有助于催化，而不是过氧化物的直接质子化。P的衰变还涉及到明显的氧同位素效应（k（16）O（2）/k（18）O（2））= 1.11 +/-0.05，表明O-O键的还原断裂是P衰变的速率控制步骤。P的衰变速率异常缓慢，这是因为该过程涉及O-O键的低（-）还原裂解，并且存在与该过程相关的大的Franck-Condon势垒。或者，O-O键的2 e（-）还原性裂解具有大得多的驱动力，其使该屏障最小化并加速该反应的速率，其与天然酶中的10（7）相似。2 e-与le-反应速率的巨大差异支持了多铜氧化酶的分子机制，其中O-2在两个2 e（-）步骤中被还原为H2O。
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 H2O. To examine the reaction between the type 2/type 3 trinuclear cluster and dioxygen, the type 1 Cu was removed and replaced with Hg2+, 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 similar to9.0 kcal/mol. The exceptionally slow rate of decay of P is explained by the fact that this process involves a le(-) 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 similar to 10(7) in the native enzyme. This large difference in rate for the 2e- versus le- process supports a molecular mechanism for multicopper oxidases in which O-2 is reduced to H2O in two 2e(-) steps.