Role of aspartate 94 in the decay of the peroxide intermediate in the multicopper oxidase Fet3p.
Role of aspartate 94 in the decay of the peroxide intermediate in the multicopper oxidase Fet3p.
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DOI：
10.1021/bi047379c
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发表时间：
2005-03
期刊：
影响因子：
2.9
通讯作者：
L. Quintanar;C. Stoj;Tzu-Pin Wang;D. Kosman;E. Solomon
中科院分区：
文献类型：
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作者：
L. Quintanar;C. Stoj;Tzu-Pin Wang;D. Kosman;E. Solomon
Fet 3 p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型位点。2型和3型中心形成一个三核簇，是O（2）还原为H（2）O的活性位点。当1型铜被耗尽（C484 S突变）时，还原的三核簇与O（2）反应生成过氧化物中间体。过氧化物中间体的衰变的动力学研究表明，羧基残基（D94在Fet 3 p）协助还原裂解的O-O键在低pH值。突变在D94残基（D94 A，D94 N，和D94 E）已进行了研究，以评估其在过氧化物中间体的衰变的作用。光谱研究表明，D94突变影响的几何和电子结构的三核簇的方式是一致的氢键连接的D94。虽然D94 E突变不影响簇与O（2）的初始反应，但D94 A突变导致更大的结构变化，使三核簇对O（2）不反应，证明D94残基的结构作用。过氧化物中间体的衰变明显受到D94 E突变的影响，证实了D94参与了该反应。D94残基似乎活化了2型Cu（+）结合水的质子以参与过渡态。这些研究为D94和质子参与O-O键的还原裂解提供了新的见解。
Fet3p is a multicopper oxidase that contains four Cu ions: one type 1, one type 2, and a coupled binuclear type 3 site. The type 2 and type 3 centers form a trinuclear cluster that is the active site for O(2) reduction to H(2)O. When the type 1 Cu is depleted (C484S mutation), the reaction of the reduced trinuclear cluster with O(2) generates a peroxide intermediate. Kinetic studies of the decay of the peroxide intermediate suggest that a carboxyl residue (D94 in Fet3p) assists the reductive cleavage of the O-O bond at low pH. Mutations at the D94 residue (D94A, D94N, and D94E) have been studied to evaluate its role in the decay of the peroxide intermediate. Spectroscopic studies show that the D94 mutations affect the geometric and electronic structure of the trinuclear cluster in a way that is consistent with the hydrogen bond connectivity of D94. While the D94E mutation does not affect the initial reaction of the cluster with O(2), the D94A mutation causes larger structural changes that render the trinuclear cluster unreactive toward O(2), demonstrating a structural role for the D94 residue. The decay of the peroxide intermediate is markedly affected by the D94E mutation, confirming the involvement of D94 in this reaction. The D94 residue appears to activate a proton of the type 2 Cu(+)-bound water for participation in the transition state. These studies provide new insight into the role of D94 and proton involvement in the reductive cleavage of the O-O bond.