Verifying the Concept of Extreme Electric Fields in Proteins using Experimentally Refined Vibrational Spectroscopic Maps

振动光谱学是一种多功能的方法，可以探索各种物理化学现象，例如静电对（生物）化学过程的贡献。描述振动-静电响应的简单框架是线性振动斯塔克效应（VSE），其使用振动探针（例如，C=O伸展）作为电场（EF）传感器在功能相关的位置，例如在催化剂活性位点。最近VSE应用的一个里程碑是直接红外（IR）光谱证据的极端活性位点EF沿着反应性C=O键在酶活性位点，降低反应的活化势垒通过静电催化的机制。对于模型酶甾酮异构酶，发现-100 cm-1的振动峰位移，这与使用线性VSE的-140 MV/cm的场一致。最近，我们报道了TEM β-内酰胺酶的更高位移和EF分别为-140 cm-1和-175 MV/cm，这是抗生素耐药性的原因。这些EF提出了一个高度相关的措施，以了解（甚至预测）抗生素耐药性的进化适应度景观或指导生物催化中从头酶的设计。然而，对线性VSE的一个批评是，它忽略了对振动峰位移的许多其他贡献，这些贡献应该被考虑用于这种极端位移和EF。这些贡献可以包括极化率、EF梯度、色散、泡利排斥、振动耦合等，并且它们的忽视可能导致不准确地确定EF和错误的结论。这项工作的目的是系统地测试极端的峰位移和EF的情况下，使用结合实验和计算的方法围绕振动光谱图（VSM）的红外光谱预测极化分子动力学（AMOEBA MD）模拟的适用性的线性VSE。VSM通过半经验或高级量子力学参数化它们与局部环境相互作用的响应来模拟振动观测。在这项工作中，VSM参数兼容的高峰位移和EF将产生的配体和酶的活性位点环境的基础上，高层次的密度泛函理论，并完善实验使用振动斯塔克光谱。在后者中，VSE在受控的外部电场中检测，从而能够直接确定静电参数作为VSM参数化中的实验约束，以实现物理上精确的参数化。VSM将用于预测来自酶/配体系统的可极化AMOEBA MD模拟的IR光谱，以（重新）解释实验峰位移。通过这种方式，我们将测试线性VSE在预测极端EF方面的可靠性，并改进我们对酶活性位点的特殊环境中的振动光谱的理解。

Vibrational spectroscopy is a versatile methodology that enables exploring various physical chemical phenomena, such as electrostatic contributions to (bio)chemical process. A simple framework to describe the vibrational-electrostatic response is the linear vibrational Stark effect (VSE), which uses vibrational probes (e.g., the C=O stretch) as electric field (EF) sensors at functionally relevant locations, such as in catalyst active sites. A recent milestone of VSE applications was the direct infrared (IR) spectroscopic evidence for extreme active site EFs along reactive C=O bonds in enzyme active sites, lowering the reaction’s activation barriers via the mechanism of electrostatic catalysis. For the model enzyme ketosteroid isomerase, vibrational peak shifts of -100 cm-1 were found, which are consistent with fields of -140 MV/cm using the linear VSE. Recently, we reported even higher shifts and EFs of -140 cm-1 and -175 MV/cm, respectively, for TEM β-lactamases, which are responsible for antibiotic resistances. These EFs present a highly relevant measure to understand (or even predict) the evolutionary fitness landscape to antibiotic resistance or to guide the design of de novo enzymes in biocatalysis. However, one criticism of the linear VSE is that it neglects many other contributions to vibrational peak shifts, that should be considered for such extreme shifts and EFs. These contributions can include polarizability, EF gradients, dispersion, Pauli repulsion, vibrational coupling, etc., and their neglect can lead to inaccurately determined EFs and to erroneous conclusions. The aim of this work is to systematically test the applicability of the linear VSE for cases of extreme peak shifts and EFs using a combined experimental and computational approach centered around vibrational spectroscopic maps (VSM) for the prediction of IR spectra from polarizable molecular dynamics (AMOEBA MD) simulations. VSMs model vibrational observables via semiempirical or high-level quantum mechanical parameterizations of their response to interactions with the local environment. In this work, VSM parameters compatible with high peak shifts and EFs will be generated for the ligands and the enzyme active site environment based on high-level density functional theory, and refined experimentally using vibrational Stark spectroscopy. In the latter, the VSE is detected in controlled, external electric fields enabling to directly determine electrostatic parameters as experimental constraints in the VSM parameterization towards physically accurate parameterizations. The VSM will be utilized to predict IR spectra from polarizable AMOEBA MD simulations of the enzyme/ligand systems in order to (re-)interpret experimental peak shifts. In this way, we will test the reliability of the linear VSE in predicting extreme EFs and refine our understanding of vibrational spectra in the peculiar environment of the enzyme active site.