title: Insights into the Proton Transfer Mechanism of a Bilin Reductase PcyA Following Neutron Crystallography.
abstract: Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IXα (BV) by phycocyanobilin:ferredoxin oxidoreductase (PcyA) via two steps of two-proton-coupled two-electron reduction. We determined the neutron structure of PcyA from cyanobacteria complexed with BV, revealing the exact location of the hydrogen atoms involved in catalysis. Notably, approximately half of the BV bound to PcyA was BVH(+), a state in which all four pyrrole nitrogen atoms were protonated. The protonation states of BV complemented the protonation of adjacent Asp105. The "axial" water molecule that interacts with the neutral pyrrole nitrogen of the A-ring was identified. His88 Nδ was protonated to form a hydrogen bond with the lactam O atom of the BV A-ring. His88 and His74 were linked by hydrogen bonds via H3O(+). These results imply that Asp105, His88, and the axial water molecule contribute to proton transfer during PcyA catalysis.
authors: †Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.|‡Graduate School of Science and Engineering, Ibaraki University, Hitachi 316-8511, Japan.
authors: Masaki
authors: M
authors: Unno
authors: †Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.|‡Graduate School of Science and Engineering, Ibaraki University, Hitachi 316-8511, Japan.|§Quantum Beam Science Center, Japan Atomic Energy Agency, Naka 319-1195, Japan.
authors: Kumiko
authors: K
authors: Ishikawa-Suto
authors: †Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.
authors: Katsuhiro
authors: K
authors: Kusaka
authors: §Quantum Beam Science Center, Japan Atomic Energy Agency, Naka 319-1195, Japan.
authors: Taro
authors: T
authors: Tamada
authors: ∥Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan.|⊥Department of Biochemistry and Applied Chemistry, National Institute of Technology, Kurume College, Kurume 830-8555, Japan.
authors: Yoshinori
authors: Y
authors: Hagiwara
authors: #Department of Medical Biochemistry, Kurume University School of Medicine, Kurume 830-0011, Japan.
authors: Masakazu
authors: M
authors: Sugishima
authors: ∇Organization for Promotion of Tenure Track, University of Miyazaki, Kiyotake 889-1692, Japan.
authors: Kei
authors: K
authors: Wada
authors: †Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.
authors: Taro
authors: T
authors: Yamada
authors: †Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.|§Quantum Beam Science Center, Japan Atomic Energy Agency, Naka 319-1195, Japan.
authors: Katsuaki
authors: K
authors: Tomoyori
authors: †Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.|‡Graduate School of Science and Engineering, Ibaraki University, Hitachi 316-8511, Japan.
authors: Takaaki
authors: T
authors: Hosoya
authors: †Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.|‡Graduate School of Science and Engineering, Ibaraki University, Hitachi 316-8511, Japan.
authors: Ichiro
authors: I
authors: Tanaka
authors: †Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.
authors: Nobuo
authors: N
authors: Niimura
authors: §Quantum Beam Science Center, Japan Atomic Energy Agency, Naka 319-1195, Japan.
authors: Ryota
authors: R
authors: Kuroki
authors: ○MARUWA Foods and Biosciences Inc., Yamatokoriyama 639-1123, Japan.
authors: Koji
authors: K
authors: Inaka
authors: †Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.
authors: Makiko
authors: M
authors: Ishihara
authors: ∥Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan.|◆Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
authors: Keiichi
authors: K
authors: Fukuyama
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001664:Biliverdine / Q000378:metabolism*; D003461:Crystallography; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D033363:Neutron Diffraction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D011522:Protons; D046939:Synechocystis / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011522:Protons; D010088:Oxidoreductases; C442236:phycocyanobilin ferredoxin oxidoreductase, Synechocystis; D001664:Biliverdine