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Primary Events in the Blue Light Sensor Plant Cryptochrome: Intraprotein Electron and Proton Transfer Revealed by Femtosecond Spectroscopy
- Immeln, Dominik, Weigel, Alexander, Kottke, Tilman, Pérez Lustres, J. Luis
- Journal of the American Chemical Society 2012 v.134 no.30 pp. 12536-12546
- Chlamydomonas reinhardtii, absorption, blue light, cations, cooling, cryptochromes, electron transfer, histidine, photoreceptors, signal transduction, spectroscopy, tryptophan
- Photoreceptors are chromoproteins that undergo fast conversion from dark to signaling states upon light absorption by the chromophore. The signaling state starts signal transduction in vivo and elicits a biological response. Therefore, photoreceptors are ideally suited for analysis of protein activation by time-resolved spectroscopy. We focus on plant cryptochromes which are blue light sensors regulating the development and daily rhythm of plants. The signaling state of these flavoproteins is the neutral radical of the flavin chromophore. It forms on the microsecond time scale after light absorption by the oxidized state. We apply here femtosecond broad-band transient absorption to early stages of signaling-state formation in a plant cryptochrome from the green alga Chlamydomonas reinhardtii. Transient spectra show (i) subpicosecond decay of flavin-stimulated emission and (ii) further decay of signal until 100 ps delay with nearly constant spectral shape. The first decay (i) monitors electron transfer from a nearby tryptophan to the flavin and occurs with a time constant of τET = 0.4 ps. The second decay (ii) is analyzed by spectral decomposition and occurs with a characteristic time constant τ₁ = 31 ps. We reason that hole transport through a tryptophan triad to the protein surface and partial deprotonation of tryptophan cation radical hide behind τ₁. These processes are probably governed by vibrational cooling. Spectral decomposition is used together with anisotropy to obtain the relative orientation of flavin and the final electron donor. This narrows the number of possible electron donors down to two tryptophans. Structural analysis suggests that a set of histidines surrounding the terminal tryptophan may act as proton acceptor and thereby stabilize the radical pair on a 100 ps time scale.