Jang Hyun-Hee, Jamakhandi Arvind P, Sullivan Shane Z, Yun Chul-Ho, Hollenberg Paul F, Miller Grover P
School of Biological Sciences and Technology and Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea.
Biochim Biophys Acta. 2010 Jun;1804(6):1285-93. doi: 10.1016/j.bbapap.2010.02.003. Epub 2010 Feb 10.
As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein-protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1*CPR complex and inactive (CYP2B1)(2)*CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge-charge interactions between CPR and complex partners.
作为一种混杂的氧化还原伙伴，细胞色素P450还原酶（CPR）的生物学作用在很大程度上取决于蛋白质-蛋白质相互作用。我们通过将D113、E115和E116突变为丙氨酸来测试一个假设的CPR对接位点，并测定其对各种电子受体的活性作为离子强度的函数。稳态细胞色素c研究表明，与野生型相比，这些突变提高了催化效率，并降低了离子强度对催化参数的影响。基于对7-乙氧基-4-三氟甲基香豆素的活性，CYP2B1和CPR倾向于形成活性CYP2B1CPR复合物和无活性的（CYP2B1）2CPR复合物，直到更高的离子强度，此时只观察到二元复合物。这些突变仅增加了二元复合物的解离常数，并抑制了离子强度效应。用非结合底物3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）进行的研究表明，对细胞色素c和CYP2B1的活性变化反映了由突变引起的电子传递途径的改变。催化和结合参数的静电建模证实了D113的重要性，特别是双突变体E115和E116作为CPR与复合物伙伴之间形成电荷-电荷相互作用的介质的重要性。
