Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions
Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions
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DOI：
10.1021/ja050321g
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发表时间：
2005-05-25
影响因子：
15
通讯作者：
Dawson, JH
中科院分区：
文献类型：
--
作者：
Fedurco, M;Augustynski, J;Dawson, JH
本文首次实验测定了未折叠金属蛋白在尿素溶液中的重组能。在9M尿素存在下，马心细胞色素c(Cytc)在pH=2时发生可逆单电子转移反应。相反，在没有尿素的低离子强度条件下，蛋白质在pH为2时电化学不活跃。研究表明，尿素可引起血红素铁的连接变化，并导致蛋白质的α-螺旋含量几乎完全丧失。尽管没有折叠，Cytc在2-巯基乙醇修饰的Ag(111)电极上的电子转移(ET)动力学仍然保持着异常快的速度和扩散控制。在9M尿素中酸滴定到pH 2时，伴随着一个轴向配体的质子化，水与血红素铁的结合(pK(A)=5.2)，以及蛋白质的突然崩溃(pH<；4)。在pH 2下，尿素展开的六配位His18-Fe(III)-H2O/五配位His18-Fe(II)对的形式氧化还原电位为-0.083 V，比双组份尿素变性的细胞色素c在pH 7时的氧化还原电位高约130 mV。在pH 2(0.4+/-0.01 eV)时，酸/尿素未折叠细胞色素c的低重组能导致了异常快速的ET动力学，这实际上低于在pH 7时的天然细胞色素c(0.6+/-0.02 eV)，但更接近于天然双组氨酸连接的Cyt b(5)(0.44+/-0.02 eV)。讨论了电子耦合和血红素平坦化对异相ET反应速率的影响。
The present investigation reports the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution. Horse heart cytochrome c (cyt c) has been found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea. In contrast, the protein is electrochemically inactive at pH 2 under low-ionic strength conditions in the absence of urea. Urea is shown to induce ligation changes at the heme iron and lead to practically complete loss of the a-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt c on a 2-mercaptoethanol-modified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt c in 9 M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (pK(a) = 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H2O/five-coordinate His18-Fe(II) couple at pH 2 is estimated to be -0.083 V vs NHE, about 130 mV more positive than seen for bis-His-ligated urea-denatured cyt c at pH 7. The unusually fast ET kinetics are assigned to low reorganization energy of acid/urea-unfolded cyt c at pH 2 (0.41 +/- 0.01 eV), which is actually lower than that of the native cyt c at pH 7 (0.6 +/- 0.02 eV), but closer to that of native bis-His-ligated cyt b(5) (0.44 +/- 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.