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A two-directional vibrational probe reveals different electric field orientations in solution and an enzyme active site

Abstract

The catalytic power of an electric field depends on its magnitude and orientation with respect to the reactive chemical species. Understanding and designing new catalysts for electrostatic catalysis thus requires methods to measure the electric field orientation and magnitude at the molecular scale. We demonstrate that electric field orientations can be extracted using a two-directional vibrational probe by exploiting the vibrational Stark effect of both the C=O and C–D stretches of a deuterated aldehyde. Combining spectroscopy with molecular dynamics and electronic structure partitioning methods, we demonstrate that, despite distinct polarities, solvents act similarly in their preference for electrostatically stabilizing large bond dipoles at the expense of destabilizing small ones. In contrast, we find that for an active-site aldehyde inhibitor of liver alcohol dehydrogenase, the electric field orientation deviates markedly from that found in solvents, which provides direct evidence for the fundamental difference between the electrostatic environment of solvents and that of a preorganized enzyme active site.

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Fig. 1: Electrostatic catalysis and the two-directional vibrational probe used in this work.
Fig. 2: Infrared absorption and Stark spectra of CXF-D at 77 K demonstrating the VSEs of the C–D (left) and C=O (right) bonds.
Fig. 3: Normalized infrared spectra of CXF-D in a series of solvents with various polarities.
Fig. 4: Construction of the field-frequency maps for C–D and C=O in the CXF-D probe using experimentally measured frequency shifts and computed electric field projections along these bonds.
Fig. 5: Comparison of the electric field orientations in solvents and at the LADH active site revealed by two-directional electric field measurements.

Data availability

The gene sequence for the wild-type enzyme in this study has been deposited in GenBank (accession code OM863576). The X-ray coordinates and structural factors have been deposited in the Protein Data Bank as entry 7RM6 for the wild-type enzyme in complex with NADH and CXF. The electronic structure partitioning methods for DFT calculations of solvent electric fields at atomic positions were implemented in the Q-Chem software package36, which is available in the 5.4.2 and later releases. Source data are provided with this paper. All the data that support the finding of this study are available within this article and its Supplementary Information and available on Figshare (https://doi.org/10.6084/m9.figshare.19248108).

Code availability

The code used for processing MD trajectories and calculating electric field projections along bond directions is available at https://github.com/YuezhiMao/2D_VSE_probe and Zenodo (https://zenodo.org/record/6300009#.Yhw0GBPMKS4)

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Acknowledgements

We express our gratitude to B. V. Plapp at the University of Iowa for providing detailed guidance on the expression and purification of LADH and much other valuable advice. We thank C.-Y. Lin for his help with X-ray crystallography, S. Schneider, J. Weaver, J. Kirsh and S. D. E. Fried for helpful discussions, T. Carver at the Stanford Nano Shared Facilities for nickel coating the Stark windows, T. McLaughlin from Stanford University Mass Spectrometry (SUMS) for technical support with the mass spectrometry. C.Z. is grateful for the Stanford Center for Molecular Analysis and Design (CMAD) Fellowship. J.K. acknowledges support from the German Research Foundation DFG-Project-ID221545957-SFB1078/B9. This work was supported in part by NIH Grant GM118044 (to S.G.B.) and the National Science Foundation grant no. CHE-1652960 (to T.E.M.). T.E.M. also acknowledges support from the Camille Dreyfus Teacher-Scholar Awards Program. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. This research also used resources of the National Energy Research Scientific Computing Center (NERSC), a US Department of Energy Office of Science User Facility operated under contract no. DE-AC02-05CH11231, and the Sherlock cluster operated by the Stanford Research Computing Center.

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Authors

Contributions

C.Z. and S.G.B. designed the research. C.Z. performed the experiments and fixed-charge MD simulations. Y.M. developed the computational protocol that combined MD simulations and QM calculations to quantify the solvent electric field contributions, implemented the electronic structure partitioning schemes in the Q-Chem software package and performed the QM calculations. J.K. performed the AMOEBA polarizable MD simulations. A.O.A. analysed the data from the MD simulations and wrote the codes to extract the truncated solute–solvent structures for the QM calculations. Z.J. synthesized N-[formyl-2H]cyclohexylformamide. C.Z., Y.M., T.E.M. and S.G.B. discussed the results and wrote the manuscript. All the authors contributed to improving the manuscript.

Corresponding authors

Correspondence to Thomas E. Markland or Steven G. Boxer.

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Nature Chemistry thanks Steven Corcelli, Jahan Dawlaty, Feng Gai, Valerie Welborn for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Materials and Methods, Supplementary Texts, Figures 1–17 and Tables 1–31.

Source data

Source Data Fig. 2

Numerical data for optical spectra.

Source Data Fig. 3

Numerical data for optical spectra.

Source Data Fig. 4

Numerical data for scatter plots.

Source Data Fig. 5

Numerical data for optical spectra and scatter plots.

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Zheng, C., Mao, Y., Kozuch, J. et al. A two-directional vibrational probe reveals different electric field orientations in solution and an enzyme active site. Nat. Chem. 14, 891–897 (2022). https://doi.org/10.1038/s41557-022-00937-w

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