title: Differentiation of photocycle characteristics of flavin-binding BLUF domains of α- and β-subunits of photoactivated adenylyl cyclase of Euglena gracilis.
abstract: Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PACα and PACβ), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PACαF2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PACβF2 domain, which like PACαF2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PACβF2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PACαF2 were 0.28-0.32 and 34-44 s. The remarkable differences between PACαF2 and PACβF2 may be related to the sensitivity of the photoactivation. In PACαF2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PACβF2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PACβF2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PACβF2 plays a main role in suppressing the quantum efficiency.
authors.affiliation: Department of Photoscience, School of Advanced Sciences, Graduate University for Advanced Studies (SOKENDAI), Shonan Village, Hayama, Kanagawa 240-0193, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Ito
authors.forename: Akio
authors.initials: A
authors.lastname: Murakami
authors.forename: Mineo
authors.initials: M
authors.lastname: Iseki
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Takahashi
authors.forename: Shoichi
authors.initials: S
authors.lastname: Higashi
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Watanabe
mesh_terms: D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D005056:Euglena gracilis / Q000201:enzymology*; D005415:Flavins / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011789:Quantum Theory; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D016415:Sequence Alignment
chemical_list: D005415:Flavins; D021122:Protein Subunits; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D000262:Adenylyl Cyclases