Lyubenova Sevdalina, Siddiqui M Khalid, Vries Marloes J M Penning de, Ludwig Bernd, Prisner Thomas F
Institute of Physical and Theoretical Chemistry, Institute of Biochemistry, Center for Biomolecular Magnetic Resonance, and Center of Excellence "Macromolecular Complexes", Johann Wolfgang Goethe University, Frankfurt am Main, Germany.
J Phys Chem B. 2007 Apr 12;111(14):3839-46. doi: 10.1021/jp065805t. Epub 2007 Mar 21.
The complex formed between cytochrome c oxidase from Paracoccus denitrificans and its electron-transfer partner cytochrome c has been studied by multi-frequency pulse electron paramagnetic resonance spectroscopy. The dipolar relaxation of a fast-relaxing paramagnetic center induced on a more slowly relaxing center can be used to measure their distance in the range of 1-4 nm. This method has been used here for the first time to study transient protein-protein complex formation, employing soluble fragments for both interacting species. We observed significantly enhanced transversal relaxation of the CuA center in cytochrome c oxidase due to the fast-relaxing iron of cytochrome c upon complex formation. The possibility to measure cytochrome c oxidase in the presence and absence of cytochrome c permitted us to separate the dipolar relaxation from other relaxation contributions. This allowed a quantitative simulation and interpretation of the relaxation data. The specific temperature dependence of the dipolar relaxation together with the high orientational selectivity achieved at high magnetic field values may provide detailed information on distance and relative orientation of the two proteins with respect to each other in the complex. Our experimental results cannot be explained by any single well-defined structure of the complex of cytochrome c oxidase with cytochrome c, but rather suggest that a broad distribution in distances and relative orientations between the two proteins exist within this complex.
利用多频脉冲电子顺磁共振光谱对反硝化副球菌细胞色素c氧化酶与其电子传递伴侣细胞色素c形成的复合物进行了研究。在一个弛豫较慢的中心上诱导产生的快速弛豫顺磁中心的偶极弛豫可用于测量它们在1-4纳米范围内的距离。本文首次使用该方法研究瞬时蛋白质-蛋白质复合物的形成，两种相互作用的物种均采用可溶性片段。我们观察到，复合物形成时，由于细胞色素c的快速弛豫铁，细胞色素c氧化酶中CuA中心的横向弛豫显著增强。在有和没有细胞色素c的情况下测量细胞色素c氧化酶的可能性使我们能够将偶极弛豫与其他弛豫贡献区分开来。这使得对弛豫数据进行定量模拟和解释成为可能。偶极弛豫的特定温度依赖性以及在高磁场值下实现的高取向选择性可能提供有关复合物中两种蛋白质彼此之间的距离和相对取向的详细信息。我们的实验结果无法用细胞色素c氧化酶与细胞色素c复合物的任何单一明确结构来解释，而是表明该复合物中两种蛋白质之间的距离和相对取向存在广泛分布。
