Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states.

Autor:	Joutsuka T; Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki, Japan; Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka-Tokai, Ibaraki, Japan. Electronic address: tatsuya.joutsuka.joe@vc.ibaraki.ac.jp., Nanasawa R; Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki, Japan., Igarashi K; Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki, Japan., Horie K; Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki, Japan., Sugishima M; Department of Medical Biochemistry, Kurume University School of Medicine, Kurume, Fukuoka, Japan., Hagiwara Y; Department of Biochemistry and Applied Chemistry, National Institute of Technology, Kurume College, Kurume, Fukuoka, Japan., Wada K; Department of Medical Sciences, University of Miyazaki, Miyazaki, Miyazaki, Japan., Fukuyama K; Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan., Yano N; Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka-Tokai, Ibaraki, Japan., Mori S; Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki, Japan; Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka-Tokai, Ibaraki, Japan., Ostermann A; Heinz Maier-Leibnitz Zentrum (MLZ), Technical University Munich, Garching, Germany., Kusaka K; Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka-Tokai, Ibaraki, Japan., Unno M; Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki, Japan; Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka-Tokai, Ibaraki, Japan. Electronic address: masaki.unno.19@vc.ibaraki.ac.jp.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2023 Jan; Vol. 299 (1), pp. 102763. Date of Electronic Publication: 2022 Dec 01.
DOI:	10.1016/j.jbc.2022.102763
Abstrakt:	PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH + ) in the wildtype (WT) PcyA-BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA-BV complex. In the previously determined absorption spectra of the PcyA D105N and I86D mutants, complexed with BV, a peak at 730 nm, observed in the WT, disappeared and increased, respectively. Here, we performed neutron crystallography and quantum chemical analysis of the D105N-BV and I86D-BV complexes to determine the protonation states of BV and the surrounding residues and study the correlation between the absorption spectra and protonation states around BV. Neutron structures elucidated that BV in the D105N mutant is in a neutral state, whereas that in the I86D mutant is dominantly in a protonated state. Glu76 and His88 showed different hydrogen bonding with surrounding residues compared with WT PcyA, further explaining why D105N and I86D have much lower activities for phycocyanobilin synthesis than the WT PcyA. Our quantum mechanics/molecular mechanics calculations of the absorption spectra showed that the spectral change in D105N arises from Glu76 deprotonation, consistent with the neutron structure. Collectively, our findings reveal more mechanistic details of bilin pigment biosynthesis. Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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