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Enzymatic X-ray absorption spectroelectrochemistry

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Abstract

Understanding the redox properties and catalytic behavior of proteins is critical for harnessing their functions in biocatalysis and to promote efficient bio-inspired catalysts design. Enzymatic X-ray absorption spectroelectrochemistry (XA-SEC) combines the insights of X-ray absorption spectroscopy with the precision of electrochemical methods to elucidate enzymes’ redox properties and catalytic behavior. Here we describe how to perform enzymatic XA-SEC experiments. The procedure begins with the preparation of the carbon-based working electrode to enhance enzyme immobilization. We exemplify with the efficient immobilization of bilirubin oxidase from Myrothecium verrucaria on the electrode surface, utilizing nanomaterials to enhance biomaterial loading and electron-transfer at the enzyme–electrode interface. Next, we guide researchers through setting up a standard three-electrode electrochemical cell, ensuring proper electrical connections and electrolyte preparation. Our Protocol details the Cu K-edge X-ray absorption spectroscopy measurement procedure at the synchrotron light sources, with in situ electrochemical control. Real-time redox processes are monitored through direct electron transfer analysis, providing valuable thermodynamic and kinetic information. It is important to determine the stability and activity of the analyzed protein under X-ray beam exposure; our approach typically results in stable electrochemical and spectroscopic signals for long experimental runs, showcasing the enzyme’s robust performance and efficient protein immobilization. The method’s ability to correlate XA-SEC data with direct electron transfer and substrate-biding analysis provides a powerful tool for advancing our understanding of enzymatic electrocatalysis and opens new avenues for developing sustainable bioelectrochemical technologies.

Key points

  • X-ray absorption spectroelectrochemistry is crucial to investigating metallic centers within metalloproteins, especially during electrochemical reactions in which redox states change.

  • Performing X-ray absorption spectroelectrochemistry involves multiple details and challenges, either from the protein, the spectroscopic or the electrochemistry sides. As synchrotron sources’ time is scarce, here, we present a Protocol with detailed steps and an extensive troubleshooting section to help researchers acquire useful data within the available time shifts.

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Fig. 1: Structural visualization of the metalloprotein and typical XAS stage/cell configuration.
Fig. 2: Overview of the Carnaúba XAS beamline; included are photographs showing the two experimental stations (Tarumã and Sapoti).
Fig. 3: A combination of techniques is essential for a deep understanding of enzymatic mechanisms, especially in the context of bioelectrocatalysis.
Fig. 4: Typical potentiostat configuration for a three-electrode experiment in XA-SEC.
Fig. 5: Representation of a CV at a nonfaradaic region.
Fig. 6: XA-SEC spectroscopic and titration graphs for ORR with MvBOD.
Fig. 7: XANES data for MvBOD during WOR.

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Data availability

All data from published works by the group are available from the corresponding author upon reasonable request.

Code availability

No code was employed.

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Acknowledgements

We thank São Paulo Research Foundation - FAPESP for the financial support, with grant nos. 2021/05665-7 (R.N.P.C.), 2018/22214-6, 2022/09164-5, 2023/01529-7 (F.N.C.) and 2020/04796-8 (G.C.S.). This research used resources from the Brazilian Synchrotron Light Laboratory (LNLS), part of the Brazilian Center for Research in Energy and Materials (CNPEM), a private nonprofit organization under the supervision of the Brazilian Ministry for Science, Technology and Innovations (MCTI). The Carnauba beamline staff are acknowledged for their assistance during the experiments.

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Authors and Affiliations

  1. São Carlos Institute of Chemistry, University of São Paulo, São Carlos, Brazil

    Rafael N. P. Colombo, Graziela C. Sedenho & Frank N. Crespilho

  2. Brazilian Synchrotron Light Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, Brazil

    Itamar T. Neckel

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  1. Rafael N. P. Colombo
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R.N.P.C., G.C.S., I.T.N. and F.N.C. contributed to writing, editing and formatting. F.N.C. provided the initial draft.

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Correspondence to Frank N. Crespilho.

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Key references

Sedenho, G. C. et al. Adv. Energy Matter. 12, 2202485 (2022): https://doi.org/10.1002/aenm.202202485

Macedo, L. J. A. et al. Nat. Commun. 11, 316 (2020): https://doi.org/10.1038/s41467-019-14210-1

Sedenho, G. C. et al. Appl. Phys. Rev. 11, 021341 (2024): https://doi.org/10.1063/5.0204996

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Colombo, R.N.P., Sedenho, G.C., Neckel, I.T. et al. Enzymatic X-ray absorption spectroelectrochemistry. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01254-5

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