Durin Guillaume, Delaunay Aude, Darnault Claudine, Heyes Derren J, Royant Antoine, Vernede Xavier, Hunter C Neil, Weik Martin, Bourgeois Dominique
Institut de Biologie Structurale Jean-Pierre Ebel, Centre d'Etudes Atomiques, Centre National de la Recherche Scientifique, Université Joseph Fourier, Grenoble, France.
Biophys J. 2009 Mar 4;96(5):1902-10. doi: 10.1016/j.bpj.2008.10.065.
Solvent fluctuations play a key role in controlling protein motions and biological function. Here, we have studied how individual steps of the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (POR) couple with solvent dynamics. To simultaneously monitor the catalytic cycle of the enzyme and the dynamical behavior of the solvent, we designed temperature-dependent UV-visible microspectrophotometry experiments, using flash-cooled nanodroplets of POR to which an exogenous soluble fluorophore was added. The formation and decay of the first two intermediates in the POR-catalyzed reaction were measured, together with the solvent glass transition and the buildup of crystalline ice at cryogenic temperatures. We find that formation of the first intermediate occurs below the glass transition temperature (T(g)), and is not affected by changes in solvent dynamics induced by modifying the glycerol content. In contrast, formation of the second intermediate occurs above T(g) and is influenced by changes in glycerol concentration in a manner remarkably similar to the buildup of crystalline ice. These results suggest that internal, nonslaved protein motions drive the first step of the POR-catalyzed reaction whereas solvent-slaved motions control the second step. We propose that the concept of solvent slaving applies to complex enzymes such as POR.
溶剂涨落在控制蛋白质运动和生物功能方面起着关键作用。在此，我们研究了光激活酶原叶绿素酸氧化还原酶（POR）催化反应的各个步骤如何与溶剂动力学相耦合。为了同时监测酶的催化循环和溶剂的动力学行为，我们设计了依赖温度的紫外可见显微分光光度法实验，使用添加了外源可溶性荧光团的POR快速冷却纳米液滴。测量了POR催化反应中前两个中间体的形成和衰减，以及溶剂玻璃化转变和低温下结晶冰的形成。我们发现第一个中间体的形成发生在玻璃化转变温度（T(g)）以下，并且不受通过改变甘油含量引起的溶剂动力学变化的影响。相比之下，第二个中间体的形成发生在T(g)以上，并且以与结晶冰形成非常相似的方式受到甘油浓度变化的影响。这些结果表明，内部的、非受迫的蛋白质运动驱动了POR催化反应的第一步，而溶剂受迫运动控制了第二步。我们提出溶剂受迫的概念适用于诸如POR这样的复杂酶。
