The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase.
The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase.
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DOI：
10.1039/d1sc01734a
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发表时间：
2021-10-13
期刊：
影响因子：
8.4
通讯作者：
Vincent KA
中科院分区：
文献类型：
--
作者：
Ash PA;Kendall-Price SET;Evans RM;Carr SB;Brasnett AR;Morra S;Rowbotham JS;Hidalgo R;Healy AJ;Cinque G;Frogley MD;Armstrong FA;Vincent KA
复杂金属酶晶体内催化相关状态的受控形成是结构-功能研究的一个重大挑战。在这里，我们展示了如何电化学控制[NiFe]氢化酶1（Hyd 1）从大肠杆菌的单晶体，使其有可能通过以前在溶液中观察到的活性位点状态的全阵列导航。电化学控制与同步辐射红外显微光谱相结合，使我们能够测量高信噪比的红外光谱原位从一个小面积的晶体。输出报告了通过氢化酶活性位点处内源性CO和CN−配体的振动伸缩带位置进行的活性位点物种形成。pH值的变化进一步证明了催化相关的质子化状态之间的平衡如何在晶体中被故意扰动，产生导致特定状态富集的电化学电势和pH条件的图。在晶体中的氧化还原滴定与测量在溶液中或电极固定的Hyd 1的比较确认的完整性的质子转移和氧化还原环境周围的活性位点的酶在晶体中。在晶体中的氢化酶中缓慢的质子转移平衡揭示了通常只能通过溶液中的超快方法观察到的转变。因此，这项研究表明了电化学控制的可能性，在稳定的特定状态的单金属酶晶体进一步研究，并扩展质子转移的[NiFe]氢化酶催化循环过程中的机械理解。单晶的电化学耦合红外显微光谱提供了深入了解质子耦合电子转移[NiFe]氢化酶。
Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure–function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1 (Hyd1) from Escherichia coli makes it possible to navigate through the full array of active site states previously observed in solution. Electrochemical control is combined with synchrotron infrared microspectroscopy, which enables us to measure high signal-to-noise IR spectra in situ from a small area of crystal. The output reports on active site speciation via the vibrational stretching band positions of the endogenous CO and CN− ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle. Electrochemically-coupled IR microspectroscopy of single crystals provides insight into proton-coupled electron transfer in [NiFe] hydrogenase.
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影响因子：
12.9
作者：
Ash PA;Hidalgo R;Vincent KA
通讯作者：
Vincent KA
影响因子：
3
作者：
Bleijlevens, B;van Broekhuizen, FA;Albracht, SPJ
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Albracht, SPJ
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15
作者：
Evans, Rhiannon M.;Ash, Philip A.;Armstrong, Fraser A.
通讯作者：
Armstrong, Fraser A.
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3
作者：
Foerster, S;van Gastel, M;Lubitz, W
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Lubitz, W
影响因子：
15
作者：
Foerster, S;Stein, M;Lubitz, W
通讯作者：
Lubitz, W