HIGH-RESOLUTION X-RAY EMISSION SPECTROSCOPY AS A PROBE OF BIO ZN SITES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。碳酸酐酶(CA)是红细胞中最常见的酶。它催化二氧化碳和碳酸之间的平衡，因此在呼吸中起着关键作用。目前已经报道了100多种CA的晶体结构，并通过各种生化和光谱探针对该酶进行了深入的研究。尽管进行了广泛的研究，但结合在锌上的溶剂分子的质子化状态的确凿证据仍然难以捉摸。最近的~(67)Zn核磁共振数据表明，锌的位置不随pH而改变，这意味着pH=6.8的转变不涉及锌结合的溶剂分子的质子化，而是一定涉及远程位置的质子化。我们去年证明，高分辨率的X射线发射可以利用配体到金属的电荷转移跃迁对局部结构的敏感性来探测锌位置的结构。我们初步报道了与锌结合溶剂质子化状态变化相一致的蛋白质。我们使用最新的束流时间以更高的信噪比重复这些测量，并证实了我们的发现，至少从X射线发射的角度来看，CA中的锌结合溶剂分子确实改变了质子化状态。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Carbonic anhydrase (CA) is the most common enzyme in red blood cells. It catalyzes the equilibration between CO2 and carbonic acid, and thus plays a critical role in respiration. Over one hundred crystals structures have been reported for CA and the enzyme has been studied in depth by a variety of biochemical and spectroscopic probes. Despite this extensive investigation, definitive evidence for the protonation state of the solvent molecule bound to the Zn has remained elusive. Recent 67Zn NMR data have suggested that the Zn site does not change with pH, implying that the pH=6.8 transition does not involve protonation of the Zn-bound solvent molecule, but rather must involve protonation at some remote site. We showed last year that high-resolution x-ray emission can be used to probe the structure of the Zn site by using the sensitivity of ligand-to-metal charge-transfer transitions to local structure. We reported preliminary protein consistent with a change in protonation state of the zinc-bound solvent. We have used the latest beam time to repeat these measurements with higher signal/noise, and have confirmed our finding that, at least from the x-ray emission perspective, the Zn-bound solvent molecule in CA does change protonation state.