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bioRxiv · 10.1101/2020.07.12.199695

Sub-3 Å resolution structure of 110 kDa nitrite reductase determined by 200 kV cryogenic electron microscopy

Naruhiko Adachi·Takahide Yamaguchi·Toshio Moriya·Masato Kawasaki·Kotaro Koiwai·Akira Shinoda·Yusuke Yamada·Fumiaki Yumoto·Takamitsu Kohzuma·Toshiya Senda
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Abstract

Cu-containing nitrite reductases (NiRs), a well-studied family of 110 kDa enzymes, play central roles in denitrification and have over 100 Protein Data Bank entries. However, such issues as crystal packing, photoreduction, and lack of high pH cases have impeded structural analysis of the catalytic mechanism. Here we show the cryogenic electron microscopy (cryo-EM) structures of Achromobacter cycloclastes NiR (AcNiR) at 2.99 and 2.85 [A] resolution with pH 6.2 and 8.1, respectively. Comprehensive comparisons with cryo-EM and 56 AcNiR crystal structures suggested crystallographic artifacts in residues 185-215 and His255 due to packing and photoreduction, respectively. With electron paramagnetic resonance spectroscopy, a newly developed map comparison method supported local structural changes at pH 8.1 around the type-2 Cu site, including His255 deprotonation. While the theoretical coordination error estimation of cryo-EM structures remains difficult, combined analysis using X-ray and cryo-EM structures will allow deeper insight into the local structural changes of proteins.

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Naruhiko Adachi, Takahide Yamaguchi, Toshio Moriya, Masato Kawasaki, Kotaro Koiwai, Akira Shinoda, Yusuke Yamada, Fumiaki Yumoto, Takamitsu Kohzuma, Toshiya Senda. 2020-07-12. Sub-3 Å resolution structure of 110 kDa nitrite reductase determined by 200 kV cryogenic electron microscopy. https://doi.org/10.1101/2020.07.12.199695

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