Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

Yohta Fukuda, Ka Man Tse, Takanori Nakane, Toru Nakatsu, Mamoru Suzuki, Michihiro Sugahara, Shigeyuki Inoue, Tetsuya Masuda, Fumiaki Yumoto, Naohiro Matsugaki, Eriko Nango, Kensuke Tono, Yasumasa Joti, Takashi Kameshima, Changyong Song, Takaki Hatsui, Makina Yabashi, Osamu Nureki, Michael E.P. Murphy, Tsuyoshi InoueSo Iwatae, Eiichi Mizohata

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- And 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redoxcoupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

Original languageEnglish
Pages (from-to)2928-2933
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number11
DOIs
Publication statusPublished - 2016 Mar 15

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography'. Together they form a unique fingerprint.

  • Cite this

    Fukuda, Y., Tse, K. M., Nakane, T., Nakatsu, T., Suzuki, M., Sugahara, M., Inoue, S., Masuda, T., Yumoto, F., Matsugaki, N., Nango, E., Tono, K., Joti, Y., Kameshima, T., Song, C., Hatsui, T., Yabashi, M., Nureki, O., Murphy, M. E. P., ... Mizohata, E. (2016). Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography. Proceedings of the National Academy of Sciences of the United States of America, 113(11), 2928-2933. https://doi.org/10.1073/pnas.1517770113