Article Decoding in-cell respiratory enzyme dynamics by label-free in situ electrochemistry

Yoshihide Tokunou ORCID ; Tomohiko Yamazaki SAMURAI ORCID ; Takashi Fujikawa ORCID ; Akihiro Okamoto SAMURAI ORCID

tokunou-et-al PNAS2025-decoding-in-cell-respiratory-enzyme-dynamics-by-label-free-in-situ-electrochemistry.pdf
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Yoshihide Tokunou, Tomohiko Yamazaki, Takashi Fujikawa, Akihiro Okamoto. Decoding in-cell respiratory enzyme dynamics by label-free in situ electrochemistry. Proceedings of the National Academy of Sciences. 2025, 122 (12), e2418926122. https://doi.org/10.1073/pnas.2418926122

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(abstract)

Deciphering metabolic enzyme catalysis in living cells remains a formidable challenge due to the limitations of in vivo assays, which focus on enzymes isolated from respiration. This study introduces an innovative whole-cell electrochemical assay to reveal the Michaelis–Menten landscape of respiratory enzymes amid complex molecular interactions. We controlled the microbial current generation’s rate-limiting step, extracting in vivo kinetic parameters (Km, Ki, and kcat) for the periplasmic nitrite (NrfA) and fumarate (FccA) reductases. Notably, while NrfA kinetics mirrored those of its purified form, FccA exhibited unique kinetic behavior. Further exploration using a mutant strain lacking CymA, a periplasmic hub protein, revealed its crucial role in modulating FccA’s kinetics, challenging the prevailing view that molecular crowding is the main cause of discrepancies between in vivo and in vitro enzyme kinetics.

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Keyword: Michaelis–Menten equation, interprotein interaction, enzymatic activity

Date published: 2025-03-25

Publisher: Proceedings of the National Academy of Sciences

Journal:

  • Proceedings of the National Academy of Sciences (ISSN: 00278424) vol. 122 issue. 12 e2418926122

Funding:

  • MEXT | JST | ACT-X JPMJAX211C
  • MEXT | Japan Society for the Promotion of Science 20K15428
  • MEXT | Japan Science and Technology Agency JPMJGX23B2
  • MEXT ARIM JPMXP1223NM5212
  • University of Tsukuba None
  • MEXT | JST | Fusion Oriented REsearch for disruptive Science and Technology JST FOREST
  • MEXT | Japan Society for the Promotion of Science 22KK0242
  • MEXT | Japan Society for the Promotion of Science 23K23532

Manuscript type: Publisher's version (Version of record)

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First published URL: https://doi.org/10.1073/pnas.2418926122

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Updated at: 2025-12-24 16:46:17 +0900

Published on MDR: 2025-12-25 08:19:49 +0900

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