Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA; Center for High Energy X-ray Sciences (CHEXS), Ithaca, New York, USA.
Center for High Energy X-ray Sciences (CHEXS), Ithaca, New York, USA.
J Biol Chem. 2023 Aug;299(8):105039. doi: 10.1016/j.jbc.2023.105039. Epub 2023 Jul 11.
Oxygen-sensitive metalloenzymes are responsible for many of the most fundamental biochemical processes in nature, from the reduction of dinitrogen in nitrogenase to the biosynthesis of photosynthetic pigments. However, biophysical characterization of such proteins under anoxic conditions can be challenging, especially at noncryogenic temperatures. In this study, we introduce the first in-line anoxic small-angle X-ray scattering (anSAXS) system at a major national synchrotron source, featuring both batch-mode and chromatography-mode capabilities. To demonstrate chromatography-coupled anSAXS, we investigated the oligomeric interconversions of the fumarate and nitrate reduction (FNR) transcription factor, which is responsible for the transcriptional response to changing oxygen conditions in the facultative anaerobe Escherichia coli. Previous work has shown that FNR contains a labile [4Fe-4S] cluster that is degraded when oxygen is present and that this change in cluster composition leads to the dissociation of the DNA-binding dimeric form. Using anSAXS, we provide the first direct structural evidence for the oxygen-induced dissociation of the E. coli FNR dimer and its correlation with cluster composition. We further demonstrate how complex FNR-DNA interactions can be studied by investigating the promoter region of the anaerobic ribonucleotide reductase genes, nrdDG, which contains tandem FNR-binding sites. By coupling size-exclusion chromatography-anSAXS with full-spectrum UV-Vis analysis, we show that the [4Fe-4S] cluster-containing dimeric form of FNR can bind to both sites in the nrdDG promoter region. The development of in-line anSAXS greatly expands the toolbox available for the study of complex metalloproteins and provides a foundation for future expansions.
氧敏感金属酶负责自然界中许多最基本的生化过程，从氮酶中还原二氮到光合色素的生物合成。然而，在缺氧条件下对这些蛋白质进行生物物理特性的表征可能具有挑战性，特别是在非低温条件下。在这项研究中，我们在一个主要的国家同步加速器源中引入了第一个在线缺氧小角 X 射线散射（anSAXS）系统，该系统具有批处理模式和色谱模式两种功能。为了演示与色谱法耦合的 anSAXS，我们研究了延胡索酸盐和硝酸盐还原（FNR）转录因子的寡聚相互转化，该因子负责响应兼性厌氧菌大肠杆菌中氧气条件变化的转录反应。以前的工作表明，FNR 含有一个不稳定的[4Fe-4S]簇，当氧气存在时，该簇会被降解，并且这种簇组成的变化导致 DNA 结合二聚体形式的解离。使用 anSAXS，我们提供了第一个直接的结构证据，证明了氧气诱导的大肠杆菌 FNR 二聚体的解离及其与簇组成的相关性。我们还进一步展示了如何通过研究厌氧核苷酸还原酶基因 nrdDG 的启动子区域来研究复杂的 FNR-DNA 相互作用，该区域包含串联的 FNR 结合位点。通过将尺寸排阻色谱-anSAXS 与全光谱紫外可见分析相结合，我们表明含有[4Fe-4S]簇的 FNR 二聚体形式可以与 nrdDG 启动子区域的两个位点结合。在线 anSAXS 的发展极大地扩展了用于研究复杂金属蛋白的工具包，并为未来的扩展提供了基础。
