title: Spectroscopic characterization of radicals and radical pairs in fruit fly cryptochrome - protonated and nonprotonated flavin radical-states.
abstract: Drosophila melanogaster cryptochrome is one of the model proteins for animal blue-light photoreceptors. Using time-resolved and steady-state optical spectroscopy, we studied the mechanism of light-induced radical-pair formation and decay, and the photoreduction of the FAD cofactor. Exact kinetics on a microsecond to minutes timescale could be extracted for the wild-type protein using global analysis. The wild-type exhibits a fast photoreduction reaction from the oxidized FAD to the FAD(•-) state with a very positive midpoint potential of ~ +125 mV, although no further reduction could be observed. We could also demonstrate that the terminal tryptophan of the conserved triad, W342, is directly involved in electron transfer; however, photoreduction could not be completely inhibited in a W342F mutant. The investigation of another mutation close to the FAD cofactor, C416N, rather unexpectedly reveals accumulation of a protonated flavin radical on a timescale of several seconds. The obtained data are critically discussed with the ones obtained from another protein, Escherichia coli photolyase, and we conclude that the amino acid opposite N(5) of the isoalloxazine moiety of FAD is able to (de)stabilize the protonated FAD radical but not to significantly modulate the kinetics of any light-inducted reactions.
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Bernd
authors: B
authors: Paulus
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Csaba
authors: C
authors: Bajzath
authors: Laboratoire de Bioélectrochimie et Spectroscopie Université de Strasbourg, France.
authors: Frédéric
authors: F
authors: Melin
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Lorenz
authors: L
authors: Heidinger
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Viktoria
authors: V
authors: Kromm
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Christoph
authors: C
authors: Herkersdorf
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Ulrike
authors: U
authors: Benz
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Lisa
authors: L
authors: Mann
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Patricia
authors: P
authors: Stehle
authors: Laboratoire de Bioélectrochimie et Spectroscopie Université de Strasbourg, France.
authors: Petra
authors: P
authors: Hellwig
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Stefan
authors: S
authors: Weber
authors: Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany.
authors: Erik
authors: E
authors: Schleicher
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D056931:Cryptochromes / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D004331:Drosophila melanogaster / Q000737:chemistry / Q000235:genetics; D004579:Electron Transport; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry / Q000528:radiation effects; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D055668:Photochemical Processes; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D013053:Spectrophotometry; D014364:Tryptophan / Q000737:chemistry
chemical_list: D056931:Cryptochromes; D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; D005136:Eye Proteins; D005609:Free Radicals; D011522:Protons; D011994:Recombinant Proteins; C119828:cry protein, Drosophila; D005182:Flavin-Adenine Dinucleotide; D014364:Tryptophan; D004255:Deoxyribodipyrimidine Photo-Lyase
keywords: blue-light photoreceptor; cryptochrome; flavoprotein; proton-transfer reaction; time resolved optical spectroscopy