Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase.
Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase.
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DOI：
10.1039/d0sc00512f
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发表时间：
2020-04-14
期刊：
影响因子：
8.4
通讯作者：
Berggren G
中科院分区：
文献类型：
--
作者：
Mészáros LS;Ceccaldi P;Lorenzi M;Redman HJ;Pfitzner E;Heberle J;Senger M;Stripp ST;Berggren G
氢酶是迄今已知的放氢最快的催化剂之一，并在体外条件下进行了广泛的研究。在这里，我们报告了第一次在全细胞条件下对[FeFe]-氢酶的机理研究。绿藻衣藻的功能性[FeFe]-氢酶是在转基因的大肠杆菌细胞中通过向生长介质中添加合成辅因子而产生的。利用全细胞电子顺磁共振、傅里叶变换红外差示光谱以及散射扫描近场光学显微镜对半合成酶的组装和反应活性进行了监测。通过气体处理、pH滴定和同位素编辑的组合，我们能够证实活细胞中一些拟议的催化中间体的形成，支持它们的生理相关性。此外，本文还报道了一种以前未完全表征的催化中间体，归因于质子化金属氢化物物种的形成。探讨了全细胞条件下[FeFe]氢酶生成氢气的机理，揭示了生理条件下活性金属氢化物物种的形成。
Hydrogenases are among the fastest H2 evolving catalysts known to date and have been extensively studied under in vitro conditions. Here, we report the first mechanistic investigation of an [FeFe]-hydrogenase under whole-cell conditions. Functional [FeFe]-hydrogenase from the green alga Chlamydomonas reinhardtii is generated in genetically modified Escherichia coli cells by addition of a synthetic cofactor to the growth medium. The assembly and reactivity of the resulting semi-synthetic enzyme was monitored using whole-cell electron paramagnetic resonance and Fourier-transform Infrared difference spectroscopy as well as scattering scanning near-field optical microscopy. Through a combination of gas treatments, pH titrations, and isotope editing we were able to corroborate the formation of a number of proposed catalytic intermediates in living cells, supporting their physiological relevance. Moreover, a previously incompletely characterized catalytic intermediate is reported herein, attributed to the formation of a protonated metal hydride species. The mechanism of hydrogen gas formation by [FeFe] hydrogenase is probed under whole cell conditions, revealing the formation of reactive metal hydride species under physiologically relevant conditions.
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