Defining the role of the axial ligand of the type 1 copper site in amicyanin by replacement of methionine with leucine.
Defining the role of the axial ligand of the type 1 copper site in amicyanin by replacement of methionine with leucine.
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DOI：
10.1021/bi900836h
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发表时间：
2009-10-06
期刊：
影响因子：
2.9
通讯作者：
Davidson VL
中科院分区：
文献类型：
--
作者：
Choi M;Sukumar N;Liu A;Davidson VL
用亮氨酸取代铜位上的甲硫氨酸轴向配体对氨兰蛋白的结构和功能的影响进行了表征。氧化和还原形式的蛋白质的晶体结构表明，铜位现在是三配位的，没有轴向配体，组氨酸提供的两种配体的铜配位距离显著增加。尽管有这些结构变化，M98L氨基花青素的吸收光谱和EPR光谱只有轻微的变化，仍然与典型的1型位点的吸收光谱和EPR光谱一致。由于M98L突变，氧化还原中点电位(Em)值变得更正127 mV，这很可能是由于铜位置的疏水性增加所致。该突变对蛋白质ET复合体中还原的M98L氨基花青素到细胞色素c-551I的电子转移(ET)反应影响最大。这一比率下降了435倍，远远超过了Em值变化的预期。对ET速率(Ket)的温度依赖性的研究表明，该突变导致反应的电子耦合(HAB)降低了13.6倍。通过对还原的M98L和天然氨基花青素的晶体结构的比较分析，预测了类似的下降。ET对该反应最直接的途径是通过Met98配体。对结构的检查表明，HAB和KET实验值大幅下降的主要决定因素是M98L氨基花青素1型位点内铜到蛋白质的距离增加。
The effects on the structure and function of amicyanin of replacing the axial methionine ligand of the type 1 copper site with leucine have been characterized. The crystal structures of the oxidized and reduced forms of the protein reveal that the copper site is now tri-coordinate with no axial ligand, and that the copper coordination distances for the two ligands provided by histidines are significantly increased. Despite these structural changes, the absorption and EPR spectra of M98L amicyanin are only slightly altered and still consistent with that of a typical type 1 site. The oxidation-reduction midpoint potential (Em) value becomes 127 mV more positive as a consequence of the M98L mutation, most likely due to increased hydrophobicity of the copper site. The most dramatic effect of the mutation was on the electron transfer (ET) reaction from reduced M98L amicyanin to cytochrome c-551i within the protein ET complex. The rate decreased 435-fold, which was much more than expected from the change in Em value. Examination of the temperature dependence of the ET rate (kET) revealed that the mutation caused a 13.6 fold decrease in the electronic coupling (HAB) for the reaction. A similar decrease was predicted from a comparative analysis of the crystal structures of reduced M98L and native amicyanins. The most direct route of ET for this reaction is through the Met98 ligand. Inspection of the structures suggests that the major determinant of the large decrease in the experimentally determined values of HAB and kET is the increased distance from the copper to the protein within the type 1 site of M98L amicyanin.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.9
作者：
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通讯作者：
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DOI：
10.1107/s090744490604947x
发表时间：
2007-02
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Bento, Isabel;Peixoto, Cristina;Zaitsev, Vjacheslav N;Lindley, Peter F
通讯作者：
Lindley, Peter F
影响因子：
2.9
作者：
Davidson, VL;Jones, LH
通讯作者：
Jones, LH
影响因子：
4.3
作者：
Davidson, VL;Jones, LH;Zhu, ZY
通讯作者：
Zhu, ZY