Bechtold R, Kuehn C, Lepre C, Isied S S
Nature. 1986;322(6076):286-8. doi: 10.1038/322286a0.
Cytochrome c can be modified by [(NH3)5RuII/III-] specifically at the imidazole moiety of histidine 33, and we have recently discussed the thermodynamics and kinetics of electron transfer within this modified protein. X-ray crystal structures of the oxidized and reduced forms of tuna cytochrome c indicate that the separation between the haem group of cytochrome c and the ruthenium label is 12-16 A. Internal electron transfer from the [(NH3)5RuII-] centre to the Fe(III) haem centre occurs with a rate constant k congruent to 53 s-1 (25 degrees C) (delta H = 3.5 kcal mol-1, delta S = -39 EU), as measured by pulse radiolysis. The measured unimolecular rate constant, k congruent to 53 s-1, is on the same timescale as a number of conformational changes that occur within the cytochrome c molecule. These results raise the question of whether electron transfer or protein conformational change is the rate limiting step in this process. We describe here an experiment that probes this intramolecular electron transfer step further. It involves reversing the direction of electron transfer by changing the redox potential of the ruthenium label. Electron transfer in the new ruthenium-cytochrome c derivative described here is from haem(II) to the Ru(III) label, whereas in (NH3)5Ru-cytochrome c the electron transfer is from Ru(II) to haem(III). Intramolecular electron transfer from haem(II) to Ru(III) in the new ruthenium-cytochrome c described here proceeds much slower (greater than 10(5) times) than the electron transfer from Ru(II) to haem(III) in the (NH3)5Ru-cytochrome c. We therefore conclude that electron transfer in cytochrome c is directional, with the protein envelope presumably involved in this directionality.
细胞色素c可被[(NH3)5RuII/III-]特异性修饰于组氨酸33的咪唑部分，我们最近讨论了这种修饰蛋白内电子转移的热力学和动力学。金枪鱼细胞色素c氧化态和还原态的X射线晶体结构表明，细胞色素c的血红素基团与钌标记之间的距离为12 - 16埃。通过脉冲辐解测量，从[(NH3)5RuII-]中心到Fe(III)血红素中心的内部电子转移速率常数k约为53 s-1（25℃）（ΔH = 3.5 kcal mol-1，ΔS = -39 EU）。测得的单分子速率常数k约为53 s-1，与细胞色素c分子内发生的一些构象变化处于同一时间尺度。这些结果提出了一个问题，即电子转移还是蛋白质构象变化是该过程中的速率限制步骤。我们在此描述了一个进一步探究这种分子内电子转移步骤的实验。它涉及通过改变钌标记的氧化还原电位来反转电子转移方向。此处描述的新钌 - 细胞色素c衍生物中的电子转移是从血红素(II)到Ru(III)标记，而在(NH3)5Ru - 细胞色素c中电子转移是从Ru(II)到血红素(III)。此处描述的新钌 - 细胞色素c中从血红素(II)到Ru(III)的分子内电子转移比(NH3)5Ru - 细胞色素c中从Ru(II)到血红素(III)的电子转移慢得多（大于10^5倍）。因此，我们得出结论，细胞色素c中的电子转移是有方向性的，蛋白质外壳可能参与了这种方向性。
