Archakov A I, Ivanov Yu D
Institute of Biomedical Chemistry, Moscow, Russia.
Biochim Biophys Acta. 2011 Jan;1814(1):102-10. doi: 10.1016/j.bbapap.2010.08.013. Epub 2010 Sep 8.
Atomic force microscopy (AFM) allows to visualize and count the individual protein molecules and their complexes within multiprotein systems. On the other hand, optical biosensor (OB) provides information on complex formation kinetics as well as complex lifetime (τ(LT)) and affinity. Comparison of complex lifetime τ(LT) with the time required for enzyme's catalytic cycle (τ(cat)) enables to characterize productive complexes and distinguish them from non-productive ones. Both these approaches were applied for the analysis of the three cytochrome P450-containing monooxygenase systems: cytochrome P450 101, cytochrome P450 11A1 and cytochrome P450 2B4. By using AFM, the formation of binary and ternary protein complexes was registered in all the three systems. OB analysis enabled to kinetically characterize these binary and ternary complexes. It was shown that the binary complexes putidaredoxin reductase (PdR)/putidaredoxin (Pd) and Pd/cytochrome P450 101 (P450 101) formed within the P450 101 system and, also, the binary complexes adrenodoxin reductase (AdR)/adrenodoxin (Ad) and Ad/cytochrome P450 11A1 (P450 11A1) formed within the P450 11A1 system are non-productive (deadlock). At the same time, the ternary PdR/Pd/P450 101 and AdR/Ad/P450 11A1 complexes proved to be productive. The binary cytochrome P450 reductase (Fp)/cytochrome P450 2B4 (2B4) complexes and the ternary Fp/2B4/cytochrome b5 (b5) complexes formed within P450 2B4 system were productive.
原子力显微镜（AFM）能够可视化并计数多蛋白系统中的单个蛋白质分子及其复合物。另一方面，光学生物传感器（OB）可提供有关复合物形成动力学以及复合物寿命（τ(LT)）和亲和力的信息。将复合物寿命τ(LT)与酶催化循环所需时间（τ(cat)）进行比较，能够对有活性的复合物进行表征，并将它们与无活性的复合物区分开来。这两种方法都被用于分析三种含细胞色素P450的单加氧酶系统：细胞色素P450 101、细胞色素P450 11A1和细胞色素P450 2B4。通过使用AFM，在所有这三种系统中都记录到了二元和三元蛋白质复合物的形成。OB分析能够从动力学角度对这些二元和三元复合物进行表征。结果表明，在P450 101系统中形成的二元复合物——恶臭假单胞菌铁氧还蛋白还原酶（PdR）/恶臭假单胞菌铁氧还蛋白（Pd）和Pd/细胞色素P450 101（P450 101），以及在P450 11A1系统中形成的二元复合物——肾上腺铁氧还蛋白还原酶（AdR）/肾上腺铁氧还蛋白（Ad）和Ad/细胞色素P450 11A1（P450 11A1）都是无活性的（停滞的）。同时，三元复合物PdR/Pd/P450 101和AdR/Ad/P450 11A1被证明是有活性的。在P450 2B4系统中形成的二元复合物——细胞色素P450还原酶（Fp）/细胞色素P450 2B4（2B4）和三元复合物Fp/2B4/细胞色素b5（b5）是有活性的。
