Transmembrane metalloenzymes encapsulating transition metal complexes are involved in electron transfer and in material transformation in living organisms. Understanding their operating principles is not only important for identifying the origin of highly efficient enzymatic reactions but also for developing sensors and catalysts inspired by metalloenzymes [1]. Protein film electrochemistry (PFE) is a powerful technique to investigate the redox behavior and enzymatic activity of transmembrane metalloenzymes even with a submonolayer amount of the target protein (~10 pmol cm–2). The challenge of PFE of metalloenzymes is the interpretation of the results obtained in PFE: PFE provides current–potential curves like cyclic voltammograms and relatively large transmembrane metalloenzymes tend to have multiple redox active cofactors inside the protein, which makes the interpretation of cyclic voltammograms difficult.We are using PFE coupled with surface-enhanced infrared absorption (SEIRA) spectroscopy, which is a surface-sensitive vibrational spectroscopy, to understand enzymatic mechanisms and protein–lipid or protein–protein interactions of transmembrane metalloenzymes at the electrode surface. In this talk, we will discuss PFE–SEIRA spectroscopy results about the determination of redox potential using a vibrational probe of carbon monoxide for heme proteins [2], the mechanistic insights into nitric oxide reduction to nitrous oxide at cytochrome c-dependent nitric oxide reductase (cNOR) using in situ PFE-SEIRA spectroscopy, and protein–lipid and protein–protein interactions with cNOR [3] and cytochrome c oxidase (CcO) [4].References.[1] M. Kato, N. Fujibayashi, D. Abe, N. Matsubara, S. Yasuda, I. Yagi, ACS Catal., 11, 2356–2365 (2021).[2] M. Kato, S. Nakagawa, T. Tosha, Y. Shiro, Y. Masuda, K. Nakata, I. Yagi, J. Phys. Chem. Lett., 9, 5196 (2018).[3] M. Kato, Y. Masuda, N. Yoshida, T. Tosha, Y. Shiro, I. Yagi, Electrochim. Acta, 373, 137888 (2021).[4] M. Kato, R. Sano, N. Yoshida, M. Iwafuji, Y. Nishiyama, S. Oka, K. Shinzawa-Itoh, Y. Nishida, Y. Shintani, I. Yagi, J. Phys. Chem. Lett., 13, 9165-9170 (2022).
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