title: Absorption and emission spectroscopic characterization of photo-dynamics of photoactivated adenylyl cyclase mutant bPAC-Y7F of Beggiatoa sp.
abstract: The photoactivated cyclase bPAC of the microbial mats bacterium Beggiatoa sp. consists of a BLUF domain and an adenylyl cyclase domain. It has strong activity of photo-induced cyclic adenylyl monophosphate (cAMP) formation and is therefore an important optogenetic tool in neuroscience applications. The SUMO-bPAC-Y7F mutant where Tyr-7 is replaced by Phe-7 in the BLUF domain has lost the typical BLUF domain photo-cycle dynamics. Instead, the investigated SUMO-bPAC-Y7F mutant consisted of three protein conformations with different triplet based photo-dynamics: (i) reversible flavin quinone (Fl) cofactor reduction to flavin semiquinone (FlH), (ii) reversible violet/near ultraviolet absorbing flavin photoproduct (FlA) formation, and (iii) irreversible red absorbing flavin photoproduct (FlC) formation. Absorption and emission spectroscopic measurements on SUMO-bPAC-Y7F were carried out before, during and after light exposure. Flavin photo-dynamics schemes are developed for the SUMO-bPAC-Y7F fractions performing photo-induced FlH, FlA, and FlC formation. Quantitative parameters of the flavin cofactor excitation, relaxation and recovery dynamics in SUMO-bPAC-Y7F are determined.
authors.affiliation: Fakultät für Physik, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany. Electronic address: alfons.penzkofer@physik.uni-regensburg.de.
authors.forename: Alfons
authors.initials: A
authors.lastname: Penzkofer
authors.affiliation: Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany.
authors.forename: Manuela
authors.initials: M
authors.lastname: Stierl
authors.affiliation: Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany; Department of Exact Sciences/Biophysics, Vrije Universiteit, De Boelelaan 1081A, 1081 HV Amsterdam, The Netherlands.
authors.forename: Tilo
authors.initials: T
authors.lastname: Mathes
authors.affiliation: Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Hegemann
mesh_terms: D065966:Absorption, Physicochemical; D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D053524:Beggiatoa / Q000201:enzymology*; D004138:Dinitrocresols / Q000378:metabolism; D004789:Enzyme Activation / Q000528:radiation effects; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D013696:Temperature
chemical_list: D004138:Dinitrocresols; C024132:4,6-dinitro-o-cresol; D000262:Adenylyl Cyclases
keywords: Anionic flavin hydoquinone charge transfer complex formation; BLUF domain; Flavin-C4a-adduct photo-cycle; Photoactivated cyclase bPAC; Soil bacterium Beggiatoa sp.; Triplet based flavin semiquinone photo-cycle