Urban Philippe, Perret Alain, Pompon Denis
TBI, CNRS, INRA, INSA, Université de Toulouse, Toulouse, France.
Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057, Evry, France.
Sci Rep. 2025 Jul 9;15(1):24584. doi: 10.1038/s41598-025-09060-5.
Cytochrome b (b) plays an important role in the enhancement of the activity of specific cytochrome P450s, especially CYP3A4. Contradictory interpretations were proposed that alternatively defend conformational or redox-based mechanisms. A semi-synthetic system involving CYP3A4, its reductase (CPR), and native or modified b5 bound to the ER membrane was used to clarify this question. This took advantage of the use of b analogs featuring cofactors with modified redox properties, in combination with rapid kinetic experiments, activity determinations, and structural predictions. The results supported a redox-based mechanism of activation in which the b5 molecules remain partially confined with specific CYP3A4 partners throughout the catalytic cycle, acting both as an electron acceptor and donor. Structural models of corresponding complexes were built using AlphaFold predictions supplemented by holoenzyme reconstruction and molecular dynamics. Results supported the formation of mutually exclusive CPR-P450 and b-P450 binary complexes, each with electron transfer capacity. The potential formation of an unusual ternary complex allowing a more delocalized electron exchange between P450 heme, FAD, FMN of CPR and b heme was also considered. Modeled formation of these alternate complexes also provided potential structural interpretations for non-redox complementary mechanisms that could contribute to the global activation mechanism of CYP3A4.
细胞色素b（b5）在增强特定细胞色素P450s（尤其是CYP3A4）的活性方面发挥着重要作用。人们提出了相互矛盾的解释，分别支持构象或基于氧化还原的机制。使用了一个半合成系统，该系统包含CYP3A4、其还原酶（CPR）以及与内质网（ER）膜结合的天然或修饰的b5，以阐明这个问题。这利用了具有修饰氧化还原特性辅因子的b5类似物，并结合快速动力学实验、活性测定和结构预测。结果支持了一种基于氧化还原的激活机制，其中b5分子在整个催化循环中与特定的CYP3A4伙伴保持部分受限，既作为电子受体又作为电子供体。使用AlphaFold预测并辅以全酶重建和分子动力学构建了相应复合物的结构模型。结果支持形成相互排斥的CPR - P450和b5 - P450二元复合物，每个都具有电子转移能力。还考虑了可能形成一种不寻常的三元复合物，该复合物允许在P450血红素、CPR的FAD、FMN和b5血红素之间进行更离域的电子交换。这些替代复合物的模拟形成也为非氧化还原互补机制提供了潜在的结构解释，这些机制可能有助于CYP3A4的整体激活机制。
