Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.
Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.
复制标题
DOI：
10.1107/s205225251800386x
复制
发表时间：
2018-05-01
期刊：
影响因子：
3.9
通讯作者：
Hough MA
中科院分区：
文献类型：
--
作者：
Horrell S;Kekilli D;Sen K;Owen RL;Dworkowski FSN;Antonyuk SV;Keal TW;Yong CW;Eady RR;Hasnain SS;Strange RW;Hough MA
MSOX（multiple serial structures from one crystal）系列晶体学实验使用受控X射线辐解和光子计数探测器进行，以确定亚硝酸铜还原酶的低剂量但高分辨率结构的序列。在190 K和室温下工作提供了更大的动态自由度，允许比在通常的100 K低温下观察更多的催化循环。 该方法证明了在可变温度下获得MSOX结构电影的潜力，从而在氧化还原酶催化过程中提供了无与伦比的结构信息水平。酶在相关氧化还原状态下的高分辨率晶体结构改变了我们对酶催化的理解。最近的发展表明，X射线可以用来，通过产生溶剂化电子，在低温（100 K）下驱动晶体中的反应，以产生酶反应的“结构电影”。 然而，在这些温度下的一个严重限制是蛋白质构象运动可以被显著抑制。在这里，最近开发的MSOX（从一个晶体的多个系列结构）的方法已被应用到亚硝酸盐结合亚硝酸铜还原酶在室温下，在190 K，接近玻璃化转变。 在这两个系列的多重结构，亚硝酸盐最初观察到的“顶帽”的几何形状，这是迅速转化为“侧上”的配置之前，转换为侧上NO，然后由NO的解离和取代水改革的静止状态。密度泛函理论计算表明，顶帽取向对应于氧化型2铜网站，而侧上的取向是一致的还原状态。它表明，在晶体内的底物到产品的转换发生在较低的辐射剂量在190 K，允许更多的酶催化循环被捕获在高分辨率比在以前的100 K实验。 在室温下，反应非常迅速，但仍有可能产生和表征几种结构状态。这些实验开辟了在多个温度下获得MSOX结构电影（MSOX-VT）的可能性，在氧化还原酶催化过程中提供了无与伦比的结构信息。
MSOX (multiple serial structures from one crystal) serial crystallography experiments were carried out using controlled X-ray radiolysis and photon-counting detectors to determine sequences of low-dose yet high-resolution structures of copper nitrite reductase. Working at 190 K and at room temperature provides greater dynamic freedom, allowing more of the catalytic cycle to be observed than at the usual cryogenic temperature of 100 K. The approach demonstrates the potential to obtain MSOX structural movies at variable temperatures, thus providing an unparalleled level of structural information during catalysis for redox enzymes. High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate ‘structural movies’ of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a ‘top-hat’ geometry, which was rapidly transformed to a ‘side-on’ configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.