title: Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography.
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 redox-coupled 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.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032;
authors.forename: Yohta
authors.initials: Y
authors.lastname: Fukuda
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan;
authors.forename: Ka Man
authors.initials: KM
authors.lastname: Tse
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;
authors.forename: Takanori
authors.initials: T
authors.lastname: Nakane
authors.affiliation: Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan; RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan;
authors.forename: Toru
authors.initials: T
authors.lastname: Nakatsu
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan; Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan;
authors.forename: Mamoru
authors.initials: M
authors.lastname: Suzuki
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan;
authors.forename: Michihiro
authors.initials: M
authors.lastname: Sugahara
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan; Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Inoue
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan;
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Masuda
authors.affiliation: Structural Biology Research Center, KEK High Energy Accelerator Research Organization, Tsukuba, Ibaraki 305-0801, Japan;
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Yumoto
authors.affiliation: Structural Biology Research Center, KEK High Energy Accelerator Research Organization, Tsukuba, Ibaraki 305-0801, Japan;
authors.forename: Naohiro
authors.initials: N
authors.lastname: Matsugaki
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan;
authors.forename: Eriko
authors.initials: E
authors.lastname: Nango
authors.affiliation: Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan;
authors.forename: Kensuke
authors.initials: K
authors.lastname: Tono
authors.affiliation: Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan;
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Joti
authors.affiliation: Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan;
authors.forename: Takashi
authors.initials: T
authors.lastname: Kameshima
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan; Department of Physics, Pohang University of Science and Technology, Pohang 790-784, Korea;
authors.forename: Changyong
authors.initials: C
authors.lastname: Song
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan;
authors.forename: Takaki
authors.initials: T
authors.lastname: Hatsui
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan;
authors.forename: Makina
authors.identifier: http://orcid.org/0000-0002-2472-1684
authors.initials: M
authors.lastname: Yabashi
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Global Research Cluster, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan;
authors.forename: Osamu
authors.initials: O
authors.lastname: Nureki
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3;
authors.forename: Michael E P
authors.initials: ME
authors.lastname: Murphy
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; inouet@chem.eng.osaka-u.ac.jp mizohata@chem.eng.osaka-u.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Inoue
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan; Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; inouet@chem.eng.osaka-u.ac.jp mizohata@chem.eng.osaka-u.ac.jp.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Mizohata
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003300:Copper / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000295:instrumentation / Q000379:methods*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D017354:Point Mutation; D011487:Protein Conformation; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D009573:Nitrites; D011522:Protons; D011993:Recombinant Fusion Proteins; D003300:Copper; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing
keywords: SAD phasing; bioinorganic chemistry; copper; damage-free structure; free electron laser