Effect of infrared and visible light on 2-azidoanthraquinone in the QA binding site of photosynthetic reaction centres. An unusual mode of activation of a photoaffinity label.
Quinone-depleted reaction centres of Rhodobacter sphaeroides were reconstituted with 2-azidoanthraquinone and irradiated with short (50 ms) pulses of intense infrared (lambda = 850 +/- 50 nm) or visible light (460 less than lambda less than 630 nm). The irradiations brought about the rapid degradation of the protein-bound photoaffinity label even though it does not absorb light in either spectral region. The decomposition of the label was accompanied by a covalent modification of subunit M and by a loss of photochemical activity of the reaction centre protein (as measured by the light-induced electron transfer onto the primary acceptor, QA). In the case of the photolysis with IR light, these effects were triggered by the reduction of the protein-bound quinone (QA) to the semiquinone (Q-A) in the process of primary charge separation. The resulting reactive species showed properties of both a semiquinone and a triplet nitrene. Efficiency and specificity of the covalent incorporation were markedly improved when visible rather than IR light was used for the photolysis, presumably, because the triplet nitrene resulting from the primary charge separation was further activated in a second light-dependent reaction. The results suggest that, in conventional photoaffinity labeling experiments, the efficiency and specificity of the covalent incorporation of an aryl azide photolabel into a target protein may be improved when the photolysis is carried out with a combination of UV and intense visible light, rather than with UV light alone.
D000345:Affinity Labels; D000880:Anthraquinones / Q000528:radiation effects*; D001386:Azides / Q000528:radiation effects*; D001426:Bacterial Proteins / Q000528:radiation effects*; D007259:Infrared Rays; D008027:Light; D010782:Photolysis; D045322:Photosynthetic Reaction Center Complex Proteins; D012242:Rhodobacter sphaeroides / Q000528:radiation effects
D000345:Affinity Labels; D000880:Anthraquinones; D001386:Azides; D001426:Bacterial Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; C051914:2-azidoanthraquinone