title: One protein, two chromophores: comparative spectroscopic characterization of 6,7-dimethyl-8-ribityllumazine and riboflavin bound to lumazine protein.
abstract: We investigated the lumazine protein from Photobacterium leiognathi in complex with its biologically active cofactor, 6,7-dimethyl-8-ribityllumazine, at different redox states and compared the results with samples containing a riboflavin cofactor. Using anaerobic photoreduction, we were able to record optical absorption kinetics from both cofactors in similar protein environments. It could be demonstrated that the protein is able to stabilize a neutral ribolumazine radical with ∼35% yield. The ribolumazine radical state was further investigated by W-band continuous-wave EPR and X-band pulsed ENDOR spectroscopy. Here, both the principal values of the g-tensor and an almost complete mapping of the proton hyperfine couplings (hfcs) could be obtained. Remarkably, the g-tensor's principal components are similar to those of the respective riboflavin-containing protein; however, the proton hfcs show noticeable differences. Comparing time-resolved optical absorption and fluorescence data from ribolumazine-containing samples, solely fluorescence but no signs of any intermediate radical or a triplet state could be identified. This is in contrast to lumazine protein samples containing the riboflavin cofactor, for which a high yield of the photogenerated triplet state and some excited flavin radical could be detected using time-resolved spectroscopy. These results clearly demonstrate that ribolumazine is a redox-active molecule and could, in principle, be employed as a cofactor in other enzymatic reactions.
authors.affiliation: Institute of Physical Chemistry, Albert-Ludwigs-University Freiburg , Albertstrasse 21, 79104 Freiburg, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Paulus
authors.forename: Boris
authors.initials: B
authors.lastname: Illarionov
authors.forename: Daniel
authors.initials: D
authors.lastname: Nohr
authors.forename: Guillaume
authors.initials: G
authors.lastname: Roellinger
authors.forename: Sylwia
authors.initials: S
authors.lastname: Kacprzak
authors.forename: Markus
authors.initials: M
authors.lastname: Fischer
authors.forename: Stefan
authors.initials: S
authors.lastname: Weber
authors.forename: Adelbert
authors.initials: A
authors.lastname: Bacher
authors.forename: Erik
authors.initials: E
authors.lastname: Schleicher
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D010776:Photobacterium / Q000378:metabolism*; D011485:Protein Binding; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012256:Riboflavin / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D011621:Pteridines; D011994:Recombinant Proteins; C001061:6,7-dimethyl-8-ribityllumazine; D012256:Riboflavin