Departamento de Química Inorgánica, Analítica y Química Física and INQUIMAE (CONICET-UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. 2, piso 1, Buenos Aires, C1428EHA, Argentina.
Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Republica, Av. Gral. Flores 2125, Montevideo, 11800, Uruguay.
Arch Biochem Biophys. 2019 Apr 15;665:96-106. doi: 10.1016/j.abb.2019.02.016. Epub 2019 Feb 25.
Here we investigated the effect of electrostatic interactions and of protein tyrosine nitration of mammalian cytochrome c on the dynamics of the so-called alkaline transition, a pH- and redox-triggered conformational change that implies replacement of the axial ligand Met80 by a Lys residue. Using a combination of electrochemical, time-resolved SERR spectroelectrochemical experiments and molecular dynamics simulations we showed that in all cases the reaction can be described in terms of a two steps minimal reaction mechanism consisting of deprotonation of a triggering group followed by ligand exchange. The pK values of the transition are strongly modulated by these perturbations, with a drastic downshift upon nitration and an important upshift upon establishing electrostatic interactions with a negatively charged model surface. The value of pK is determined by the interplay between the acidity of a triggering group and the kinetic constants for the forward and backward ligand exchange processes. Nitration of Tyr74 results in a change of the triggering group from Lys73 in WT Cyt to Tyr74 in the nitrated protein, which dominates the pK downshift towards physiological values. Electrostatic interactions, on the other hand, result in strong acceleration of the backward ligand exchange reaction, which dominates the pK upshift. The different physicochemical conditions found here to influence pK are expected to vary depending on cellular conditions and subcellular localization of the protein, thus determining the existence of alternative conformations of Cyt in vivo.
在这里，我们研究了静电相互作用和哺乳动物细胞色素 c 的酪氨酸残基硝化对所谓的碱性转变的动力学的影响，碱性转变是一种 pH 和氧化还原触发的构象变化，涉及轴向配体 Met80 被 Lys 残基取代。我们使用电化学、时间分辨 SERR 光谱电化学实验和分子动力学模拟的组合表明，在所有情况下，反应都可以用一个两步最小反应机制来描述，该机制由引发基团的去质子化和配体交换组成。这些扰动强烈调节了转变的 pK 值，硝化后会急剧下降，与带负电荷的模型表面建立静电相互作用后会显著上升。pK 值取决于触发基团的酸度和正向和反向配体交换过程的动力学常数之间的相互作用。Tyr74 的硝化导致从 WT Cyt 中的 Lys73 到硝化蛋白中的 Tyr74 的触发基团的变化，这主导了向生理值的 pK 急剧下降。另一方面，静电相互作用导致反向配体交换反应的强烈加速，这主导了 pK 的上升。这里发现影响 pK 的不同物理化学条件预计会根据细胞条件和蛋白质的亚细胞定位而变化，从而决定了 Cyt 在体内存在替代构象。
