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The role of molecular crowding in long-range metalloprotein electron transfer: Dissection into site- and scaffold-specific contributions
Electrochimica Acta ( IF 5.5 ) Pub Date : 2018-10-16 , DOI: 10.1016/j.electacta.2018.10.069
Ulises A. Zitare , Jonathan Szuster , Magali F. Scocozza , Andrés Espinoza-Cara , Alcides J. Leguto , Marcos N. Morgada , Alejandro J. Vila , Daniel H. Murgida

Here we report the effect of molecular crowding on long-range protein electron transfer (ET) and disentangle the specific responses of the redox site and the protein milieu. To this end, we studied two different one-electron redox proteins that share the cupredoxin fold but differ in the metal center, T1 mononuclear blue copper and binuclear CuA, and generated chimeras with hybrid properties by incorporating different T1 centers within the CuA scaffold or by swapping loops between orthologous proteins from different organisms to perturb the CuA site. The heterogeneous ET kinetics of the different proteins was studied by protein film electrochemistry at variable electronic couplings and in the presence of two different crowding agents. The results reveal a strong frictional control of the ET reactions, which for 10 Å tunnelling distances results in a 90% drop of the ET rate when viscosity is matched to that of the mitochondrial interior (ca. 55 cP) by addition of either crowding agent. The effect is ascribed to the dynamical coupling of the metal site and the milieu, which for T1 is found to be twice stronger than for CuA, and the activation energy of protein-solvent motion that is dictated by the overall scaffold. This work highlights the need of explicitly considering molecular crowding effects in protein ET.



中文翻译:

分子拥挤在远距离金属蛋白电子转移中的作用:解剖成特定于位点和支架的贡献

在这里,我们报告了分子拥挤对远程蛋白质电子转移(ET)的影响,并弄清了氧化还原位点和蛋白质环境的特异性反应。为此,我们研究了两种不同的单电子氧化还原蛋白,它们共享铜氧还蛋白折叠,但在金属中心不同,T1单核蓝铜和双核Cu A,并通过在Cu A支架中掺入不同的T1中心生成具有杂合特性的嵌合体。或通过交换来自不同生物体的直系同源蛋白之间的环来扰动Cu A地点。通过蛋白质膜电化学在可变电子偶联和存在两种不同拥挤剂的情况下研究了不同蛋白质的异质ET动力学。结果表明,ET反应具有很强的摩擦控制能力,通过添加任一种拥挤剂,当粘度与线粒体内部的粘度(约55 cP)相匹配时,在10隧穿距离下,ET速率降低90%。 。这种作用归因于金属位点和环境的动态耦合,发现T1的强度比Cu A的强度强两倍,并且蛋白质-溶剂运动的活化能由整个支架决定。这项工作强调了明确考虑蛋白质ET中分子拥挤效应的必要性。

更新日期:2018-10-16
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