德国柏林工业大学Ingo Zebger课题组近日通过一种针对溶剂化、冻干化和晶体化金属酶的先进实验手段，研究了镍铁氢化酶氧化还原中间体的结构和功能。2021年3月30日出版的《德国应用化学》发表了这项成果。

对复杂金属酶的深刻机理表征需要多种互补技术。在这种背景下，该课题组开发了一种新的实验装置，可以对催化中间体进行可控制备，以通过各种光谱学技术对其进行表征。此外，对于气体交换时酶的冻干粉、晶体或溶液，还可以在广泛的温度范围内通过红外光谱原位监测其氧化还原转变。

作为这一概念的证明，研究人员用两种耐氧气的镍铁氢化酶作为模型系统进行研究。首先，课题组利用他们的平台制备了处于顺磁态且含有桥氢的高浓度氢化酶冻干粉。这一过程被证明有利于⁵⁷Fe核共振振动光谱，结合密度泛函理论计算，该催化中间体的振动指纹得到了揭示。

此外，结合共振拉曼光谱，同样的原位红外装置对酶晶体的氧化还原化学提供了详细的了解，这再一次说明了用光谱学分析补充X射线晶体学数据的普遍必要性。

附：英文原文

Title: Exploring structure and function of redox intermediates of [NiFe]‐hydrogenases by an advanced experimental approach for solvated, lyophilized and crystallized metalloenzymes

Author: Christian Lorent, Vladimir Pelmenschikov, Stefan Frielingsdorf, Janna Schoknecht, Giorgio Caserta, Yoshitaka Yoda, Hongxin Wang, Kenji Tamasaku, Oliver Lenz, Stephen P. Cramer, Marius Horch, Lars Lauterbach, Ingo Zebger

Issue&Volume: 2021-03-30

Abstract: A profound mechanistic characterization of complex metalloenzymes requires a multitude of complementary techniques. In this context, we developed a new experimental setup allowing controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The  in situ  monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals and solution during gas exchange in a wide temperature range can be accomplished as well. As a proof of concept, two O  2  ‐tolerant [NiFe]‐hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for  57  Fe nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. Furthermore, in combination with resonance Raman spectroscopy, the same  in situ  IR setup provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X‐ray crystallographic data with spectroscopic analyses.

DOI: 10.1002/anie.202100451

Source: https://onlinelibrary.wiley.com/doi/10.1002/anie.202100451