Hedison Tobias M, Hay Sam, Scrutton Nigel S
Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, UK.
FEBS J. 2015 Nov;282(22):4357-75. doi: 10.1111/febs.13501. Epub 2015 Sep 16.
Protein domain dynamics and electron transfer chemistry are often associated, but real-time analysis of domain motion in enzyme-catalysed reactions and the elucidation of mechanistic schemes that relate these motions to the reaction chemistry are major challenges for biological catalysis research. Previously we suggested that reduction of human cytochrome P450 reductase with the reducing coenzyme NADPH is accompanied by major structural re-orientation of the FMN- and FAD-binding domains through an inferred dynamic cycle of 'open' and 'closed' conformations of the enzyme (PLoS Biol, 2011, e1001222). However, these studies were restricted to stopped-flow/FRET analysis of the reductive half-reaction, and were compromised by fluorescence quenching of the acceptor by the flavin cofactors. Here we have improved the design of the FRET system, by using dye pairs with near-IR fluorescence, and extended studies on human cytochrome P450 reductase to the oxidative half-reaction using a double-mixing stopped-flow assay, thereby analysing in real-time conformational dynamics throughout the complete catalytic cycle. We correlate redox changes accompanying the reaction chemistry with protein dynamic changes observed by FRET, and show that redox chemistry drives a major re-orientation of the protein domains in both the reductive and oxidative half-reactions. Our studies using the tractable (soluble) surrogate electron acceptor cytochrome c provide a framework for analysing mechanisms of electron transfer in the endoplasmic reticulum between cytochrome P450 reductase and cognate P450 enzymes. More generally, our work emphasizes the importance of protein dynamics in intra- and inter-protein electron transfer, and establishes methodology for real-time analysis of structural changes throughout the catalytic cycle of complex redox proteins.
蛋白质结构域动力学与电子转移化学常常相互关联，但对酶催化反应中结构域运动进行实时分析以及阐明将这些运动与反应化学联系起来的机制方案，是生物催化研究面临的重大挑战。此前我们提出，用还原型辅酶NADPH还原人细胞色素P450还原酶时，FMN和FAD结合结构域会通过推测的酶“开放”和“关闭”构象的动态循环发生主要的结构重新定向（《公共科学图书馆·生物学》，2011年，e1001222）。然而，这些研究仅限于对还原半反应进行停流/FRET分析，并且受到黄素辅因子对受体荧光猝灭的影响。在此，我们通过使用具有近红外荧光的染料对改进了FRET系统的设计，并利用双混合停流测定法将对人细胞色素P450还原酶的研究扩展到氧化半反应，从而实时分析整个催化循环中的构象动力学。我们将反应化学过程中伴随的氧化还原变化与通过FRET观察到的蛋白质动态变化相关联，结果表明氧化还原化学在还原和氧化半反应中都驱动了蛋白质结构域的主要重新定向。我们使用易于处理的（可溶性）替代电子受体细胞色素c进行的研究，为分析内质网中细胞色素P450还原酶与同源P450酶之间的电子转移机制提供了一个框架。更广泛地说，我们的工作强调了蛋白质动力学在蛋白质内和蛋白质间电子转移中的重要性，并建立了在复杂氧化还原蛋白质的整个催化循环中实时分析结构变化的方法。
