Primary Events in the Blue Light Sensor Plant Cryptochrome: Intraprotein Electron and Proton Transfer Revealed by Femtosecond Spectroscopy

Abstract

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 τ(ET) = 0.4 ps. The second decay (ii) is analyzed by spectral decomposition and occurs with a characteristic time constant τ(1) = 31 ps. We reason that hole transport through a tryptophan triad to the protein surface and partial deprotonation of tryptophan cation radical hide behind τ(1). 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.

Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome

“…Type I Cryptochromes-The decisive steps of signal generation in the plant cryptochrome between the initial ultrafast electron transfer to FAD (14) and the structural change in the CCT (24,25) are unclear. In particular, the role of the proton transfer to the flavin radical needs to be elucidated considering the fact that the aspartic acid close to flavin is exclusively present in the plant cryptochrome.…”

“…Much less is known about the role of the subsequent step of proton transfer to flavin. In plant cryptochromes, this step is unusually separated in time from the initiating electron transfer by 6 orders of magnitude (14,15). A much faster proton transfer has been postulated in theoretical studies (31), but indications for such a competing process have been found only to some extent above the physiological pH (pH 7.4) and in the absence of ATP (32).…”

“…Upon illumination of plant cryptochromes, the oxidized FAD is reduced to the anion radical by a nearby tryptophan residue with a time constant of 400 femtoseconds (14) and subsequently protonated within a few microseconds (15,16) to form the neutral radical of FAD (17) (Fig. 1).…”

“…CPH1 groups together with cryptochromes from higher plants, mosses, and ferns to the subfamily plant cryptochrome (1). Accordingly, CPH1-PHR exhibits all characteristics of PHRs from higher plant cryptochromes (14,15,23,39). We exchanged aspartic acid with cysteine (D393C) and found that proton transfer is completely blocked in this mutant but that conformational changes are retained.…”

Ascorbic acid may not be involved in cryptochrome-based magnetoreception

“…•+ ], on the other hand, can persist for as long as microseconds [1,[12][13][14] -long enough to allow the yield of the signalling state to depend on the direction of an Earth-strength magnetic field. The identity of this long-lived form of the protein is uncertain; the assumption is that it passes on magnetic information via a conformational change in the C-terminal domain [15].…”