Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsenvej 40, DK-1871 Frederiksberg C, Denmark.
ACS Chem Biol. 2014 Mar 21;9(3):630-4. doi: 10.1021/cb400708v. Epub 2014 Jan 17.
Electron transfer between membrane spanning oxidoreductase enzymes controls vital metabolic processes. Here we studied for the first time with single molecule resolution the function of P450 oxidoreductase (POR), the canonical membrane spanning activator of all microsomal cytochrome P450 enzymes. Measurements and statistical analysis of individual catalytic turnover cycles shows POR to sample at least two major functional states. This phenotype may underlie regulatory interactions with different cytochromes P450 but to date has remained masked in bulk kinetics. To ensure that we measured the inherent behavior of POR, we reconstituted the full length POR in "native like" membrane patches, nanodiscs. Nanodisc reconstitution increased stability by ∼2-fold as compared to detergent solubilized POR and showed significantly increased activity at biologically relevant ionic strength conditions, highlighting the importance of studying POR function in a membrane environment. This assay paves the way for studying the function of additional membrane spanning oxidoreductases with single molecule resolution.
电子在跨膜氧化还原酶之间的转移控制着重要的代谢过程。在这里，我们首次以单分子分辨率研究了 P450 氧化还原酶（POR）的功能，POR 是所有细胞色素 P450 酶的典型跨膜激活剂。对单个催化周转循环的测量和统计分析表明，POR 至少可以采样两种主要的功能状态。这种表型可能是与不同细胞色素 P450 相互作用的基础，但迄今为止，在批量动力学中仍然被掩盖。为了确保我们测量的是 POR 的固有行为，我们在“类似天然”的膜片中重新构建了全长 POR，即纳米盘。与去污剂溶解的 POR 相比，纳米盘重建使 POR 的稳定性提高了约 2 倍，并且在生物学相关的离子强度条件下显示出显著增加的活性，这突出了在膜环境中研究 POR 功能的重要性。该测定方法为使用单分子分辨率研究其他跨膜氧化还原酶的功能铺平了道路。
