Institute of Microbiology, Department of Applied and Ecological Microbiology, Friedrich Schiller University Jena, Jena, Germany.
Protein Sci. 2019 Oct;28(10):1902-1908. doi: 10.1002/pro.3699. Epub 2019 Aug 7.
Corrinoids are essential cofactors of enzymes involved in the C metabolism of anaerobes. The active, super-reduced [Co ] state of the corrinoid cofactor is highly sensitive to autoxidation. In O-demethylases, the oxidation to inactive [Co ] is reversed by an ATP-dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)-diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [Co ]/[Co ] couple of the protein-bound corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the corrinoid as the electron-accepting site is achieved by increasing the potential of the corrinoid cofactor from -530 ± 15 mV to -250 ± 10 mV (E , pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide-bound AE with the corrinoid protein or its cofactor. The remaining 150-200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)-DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low-potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.
类咕啉是参与厌氧菌 C 代谢的酶的必需辅助因子。类咕啉辅因子的活性、超还原 [Co ] 状态对自动氧化极为敏感。在 O-去甲基酶中，通过激活酶 (AE) 催化的依赖 ATP 的电子转移将氧化为非活性 [Co ] 逆转。通过与紫外可见光谱耦合的电位滴定研究了在该反应过程中类咕啉辅因子发生的氧化还原电位变化。通过应用铕 (II)-二乙三胺五乙酸 (DTPA) 作为还原剂，我们能够确定在不存在和存在 AE 和/或 ATP 的情况下，蛋白质结合的类咕啉辅因子的 [Co ]/[Co ] 偶的中点电位。数据表明，通过将电子从生理供体转移到作为电子接受部位的类咕啉，类咕啉辅因子的电位从-530±15 mV 增加到-250±10 mV (E, pH 7.5)。氧化还原电位的前 50 至 100 mV 的偏移似乎是由核苷酸结合的 AE 与类咕啉蛋白或其辅因子的相互作用引起的。剩余的 150-200 mV 必须由 ATP 水解的化学能克服。实验表明，Eu(II)-DTPA 已经是一种强大的还原剂，是用于低电位氧化还原中心滴定实验的合适电子供体。此外，这项研究的结果将有助于理解由 ATP 水解的动力驱动的热力学不利的电子转移过程。
