The study of the structure, function, folding and conformational transitions of cytochrome c is of great interest because this protein plays an important role in biological electron transport and apoptosis. The different native and non-native conformations have been studied extensively under equilibrium conditions at different pH values, however, kinetic studies are rare because they require technically challenging rapid mixing and spectroscopic monitoring techniques. Here we present the refolding kinetics of acid denatured cytochrome c using the pH jump technique from pH 2 to pH 4.7 in combination with a new ultrafast continuous flow mixing device that allows time resolved measurements to the microsecond time scale. Our results show that the initial refolding of denatured oxidized cytochrome c occurs very rapidly with a time constant τ = 10 μs, and is followed by discrete refolding steps with time constants of 56 and 208 μs. Electron paramagnetic resonance analysis of the different intermediates, obtained by microsecond freeze hyper quenching showed that the first two intermediates are predominantly high spin, and the third intermediate is the low spin species with complete His/Met coordination. The initial rapid phase is characterized by the formation of high spin species distinct from the completely unfolded state. We interpret this as the formation of a five coordinate species with His18 as the axial ligand or six coordinate with water and His18 as the axial ligands.

细胞色素c的结构，功能，折叠和构象转变的研究引起了极大的兴趣，因为这种蛋白质在生物电子运输和凋亡中起着重要的作用。已经在平衡条件下在不同pH值下对不同的天然和非天然构象进行了广泛的研究，然而，动力学研究很少，因为它们需要技术上具有挑战性的快速混合和光谱监测技术。在这里，我们介绍了酸性变性细胞色素c的重折叠动力学结合使用从pH 2到pH 4.7的pH跳跃技术以及新型超快连续流混合设备，该设备可将时间分辨的测量值扩展到微秒级。我们的结果表明，变性的氧化细胞色素c的初始重折叠时间常数τ= 10μs时会非常迅速地发生，随后是时间常数分别为56和208μs的离散重折叠步骤。通过微秒冷冻超淬火获得的不同中间体的电子顺磁共振分析表明，前两个中间体主要是高自旋，而第三个中间体是具有完全的His / Met配位的低自旋物种。初始快速阶段的特征是形成了与完全展开状态不同的高自旋物种。我们将其解释为以His18为轴向配体的五个配位物种或以水和His18为轴向配体的六个配位物种的形成。