The role of acidic residues of plastocyanin in its interaction with cytochrome f
The role of acidic residues of plastocyanin in its interaction with cytochrome f
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DOI：
10.1016/s0005-2728(96)00090-4
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发表时间：
1996-11-12
影响因子：
4.3
通讯作者：
Bendall, DS
中科院分区：
文献类型：
--
作者：
Kannt, A;Young, S;Bendall, DS
菠菜质体蓝素的酸性补丁的相互作用与可溶性形式的芜菁细胞色素f的作用进行了研究相结合的定点诱变，NMR光谱和动力学分析。两个“东部”补丁，分别由保守的酸性残基42-45和59-61组成的电荷，被改变的中性或带正电荷的基团的掺入。在6个不同的突变体中缺失了多达4个负电荷，另一个突变体Q88 E在同一区域提供了额外的负电荷。采用停流分光光度法测定细胞色素f还原的总体二级速率常数（k（2））。对于每个被消除的负电荷，无论其位置如何，都观察到k（2）降低2至3倍，并且在Q88 E中有20%的增加。从离子强度依赖性的k（2）在无限的离子强度预测的天然和突变体蛋白质的类似值，而静电引力项减少与每个负电荷删除。结合平衡常数（K-A）由质体蓝素H-1共振T-2的变化确定。负电荷的丢失导致K-A的显著降低，与k（2）的降低大致平行，这表明主要影响是结合而不是复合物内电子转移的速率。两者合计，这些结果提供了令人信服的证据参与的两个酸性补丁的残基与细胞色素f的相互作用。
The role of the acidic patches of spinach plastocyanin in the interaction with a soluble form of turnip cytochrome f was studied by a combination of site-directed mutagenesis, NMR spectroscopy and kinetic analysis. The charge of the two 'eastern' patches, consisting of conserved acidic residues 42-45 and 59-61 respectively, was altered by incorporation of neutral or positively charged groups. Up to four negative charges were deleted in six different mutants and a further mutant, Q88E, provided an additional negative charge in the same region. Overall second-order rate constants (k(2)) for reduction by cytochrome f were determined by stopped-flow spectrophotometry. A 2- to 3-fold decrease in k(2) was observed for each negative charge abolished, regardless of its position, and in Q88E there was a 20% increase. From the ionic strength dependence similar values for k(2) at infinite ionic strength were predicted for the native and mutant proteins, while the electrostatic attraction term decreased with each negative charge removed. The equilibrium constant for association (K-A) was determined from the change in T-2 of H-1 resonances of plastocyanin. Loss of negative charges caused marked decreases in K-A roughly in parallel with the decreases in k(2), which suggests that the main effect was on binding rather than the rate of intracomplex electron transfer. Taken together, these results provide convincing evidence for participation of residues of both acidic patches in the interaction with cytochrome f.