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

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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.