Fukuda Yohta, Tse Ka Man, Suzuki Mamoru, Diederichs Kay, Hirata Kunio, Nakane Takanori, Sugahara Michihiro, Nango Eriko, Tono Kensuke, Joti Yasumasa, Kameshima Takashi, Song Changyong, Hatsui Takaki, Yabashi Makina, Nureki Osamu, Matsumura Hiroyoshi, Inoue Tsuyoshi, Iwata So, Mizohata Eiichi
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan;
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan; RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan;
J Biochem. 2016 May;159(5):527-38. doi: 10.1093/jb/mvv133. Epub 2016 Jan 14.
Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR.
连续飞秒晶体学（SFX）已能够对金属酶进行无损结构测定，并填补了我们在晶体学数据和光谱数据之间的知识空白。然而，晶体学家几乎不知道这种新兴技术是否能为金属酶的作用机制提供真正全新的结构见解，部分原因是分辨率有限。亚硝酸铜还原酶（CuNiR）在反硝化作用中将亚硝酸盐转化为一氧化氮，已通过同步辐射晶体学（SRX）进行了广泛研究。尽管有人怀疑CuNiR的催化铜（2型铜（T2Cu））能耐受X射线光还原作用，但我们在此表明，在SRX中，不含亚硝酸盐形式的T2Cu会被还原并改变其配位结构。此外，我们利用SFX在1.43 Å分辨率下确定了完全氧化的CuNiR结构。高分辨率的SFX数据与SRX数据的比较揭示了X射线光还原作用导致催化组氨酸残基发生的细微结构变化。这一SRX未能发现的发现为CuNiR的反应机制提供了新的见解。
