Irreversible photoreduction of flavin in a mutated Phot-LOV1 domain.
Phot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN). Upon absorption of blue light, the LOV domains undergo a photocycle, transiently forming a covalent adduct of a cysteine residue and the FMN (LOV-390). The mechanism of formation of this flavin-thiol adduct is still unclear. We studied a mutant of the LOV1 domain from the green alga Chlamydomonas reinhardtii with a methionine replacing the reactive cysteine 57 (C57M). As in the wild type, irradiation leads to formation of a photoadduct, which, however, is irreversibly converted into a red absorbing species, C57M-675. On the basis of spectroscopic results and the 2.1 A resolution crystal structure, this highly unusual FMN species was assigned to a neutral flavin radical covalently attached to the apoprotein at the N(5) position. In contrast to other flavoprotein neutral radicals, C57M-675 is stable even under aerobic or denaturing conditions. Pathways for the photoinduced formation of the adduct are discussed for the C57M mutant as well as the wild-type LOV1 domain.
Kottke
Tilman
T
Institut für Physikalische und Theoretische Chemie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany. tilman.kottke@chemie.uni-regensburg.de
Dick
Bernhard
B
Fedorov
Roman
R
Schlichting
Ilme
I
Deutzmann
Rainer
R
Hegemann
Peter
P
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D003624:Darkness; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000528:radiation effects; D005420:Flavoproteins / Q000737:chemistry* / Q000502:physiology / Q000528:radiation effects; D008027:Light; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction / Q000528:radiation effects; D010750:Phosphoproteins / Q000737:chemistry* / Q000502:physiology / Q000528:radiation effects; D010777:Photochemistry / Q000379:methods; D017956:Photoreceptor Cells, Invertebrate / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
D005420:Flavoproteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D005486:Flavin Mononucleotide; D008715:Methionine; D003545:Cysteine