Two protonation states and structural features of a bilin reductase PcyA revealed by neutron crystallography

Unno, Masayoshi*; Sugishima, Masakazu*; Wada, Kei*; Hagiwara, Yoshinori*; Kusaka, Katsuhiro*; Tamada, Taro; Fukuyama, Keiichi*

Bilin compounds are fundamentally important for oxygenic photosynthetic organisms, because they are utilized as pigments for photosynthesis (phycobilins) and photoreceptors (phytochromobilin). Phycocyanobilin (PCB), a phycobilin, comprises the chromophore of algal phytochromes and the core phycobiliprotein antennae of cyanobacteria and red algae. PCB is biosynthesized by a member of the ferredoxin-dependent bilin reductase family, phycocyanobilin:ferredoxin oxidoreductase (PcyA). In the present study, we determined the neutron crystal structure of PcyA in complex with its substrate biliverdin (BV). This neutron structure revealed the protonation state of BV and the surrounding residues. We found that two forms of BV, neutral BV and protonated BVH, were coupled with the two conformation/protonation states of the essential residue Asp105. Further, His88 and His74 near BV were singly protonated and were connected with an intervening hydronium ion. Neutron analysis also revealed how X-ray irradiation of the PcyA-BV crystal altered the structure of the PcyA-BV complex.