Structural studies of two mutants of amicyanin from Paracoccus denitrificans that stabilize the reduced state of the copper
Structural studies of two mutants of amicyanin from Paracoccus denitrificans that stabilize the reduced state of the copper
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DOI：
10.1021/bi049634z
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发表时间：
2004-07-27
期刊：
影响因子：
2.9
通讯作者：
Mathews, FS
中科院分区：
文献类型：
--
作者：
Carrell, CJ;Sun, DP;Mathews, FS
Pro94突变为苯丙氨酸或丙氨酸显著改变氨基花青素的I型铜中心的氧化还原性质。每个突变都会增加氧化还原中点电位(E)。E-m值至少降低140 mV，并将E-m值的pH依赖性的pK(A)移至更酸性的值。测定了P94F和P94A氨基花青素在氧化和还原状态下的原子分辨(0.99-1.1埃)结构。在每个氨基蓝蛋白突变体中，通过Phe94和Ala94的酰胺氮的移动，在Cys92的铜配位的硫代硫上引入了一个吸电子氢键，比野生型的氨基蓝蛋白更接近硫代硫。这可能是这些突变导致的E-m值更正的可能原因。在突变体中观察到的E-m值的pH依赖性的pK(A)值的下降似乎与突变导致的His95铜配体旋转的空间位阻有关。在野生型氨基蓝蛋白中，His95侧链经历了氧化还原和pH依赖性的构象变化，这解释了氨基蓝蛋白的E-m值的pH依赖性。还原的P94A氨基花青素呈现两种交替的构象，铜的位置相隔1.4埃。在其中一种构象中，水分子似乎取代了金属98成为铜配体。讨论了这些结构与P94F和P94A氨基花青素的电子转移性质之间的关系。
Mutation of Pro94 to phenylalanine or alanine significantly alters the redox properties of the type I copper center of amicyanin. Each mutation increases the redox midpoint potential (E.) value by at least 140 mV and shifts the pK(a) for the pH dependence of the E-m value to a more acidic value. Atomic resolution (0.99-1.1 Angstrom) structures of both the P94F and P94A amicyanin have been determined in the oxidized and reduced states. In each amicyanin mutant, an electron-withdrawing hydrogen bond to the copper-coordinating thiolate sulfur of Cys92 is introduced by movement of the amide nitrogens of Phe94 and Ala94 much closer to the thiolate sulfur than in wild-type amicyanin. This is the likely explanation for the much more positive E-m values which result from each of these mutations. The observed decrease in the pK(a) value for the pH dependence of the E-m value that is seen in the mutants seems to be correlated with steric hindrance to the rotation of the His95 copper ligand which results from the mutations. In wild-type amicyanin the His95 side chain undergoes a redox and pH-dependent conformational change which accounts for the pH dependence of the E-m value of amicyanin. The reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 Angstrom apart. In one of these conformations, a water molecule appears to have replaced Met98 as a copper ligand. The relevance of these structures to the electron transfer properties of P94F and P94A amicyanin are also discussed.