ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch

Abstract

Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH· radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·−, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396−. Its negative charge could trigger conformational changes necessary for signal transduction.

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

“…Deprotonation of this residue accompanies flavin reduction, as has been deduced from FTIR spectroscopic experiments and structural considerations (23,35,36). In support of this finding, a high pK a value of this aspartic acid has been indirectly determined by its impact on the absorption spectrum of the flavin (32). According to this study, it shifts strongly from 3.7 in solution to 7.4 in AtCRY1 and to above 9 upon addition of ATP.…”

“…Recent experimental and theoretical studies on AtCRY1 rationalized the higher yield of radical formation in the presence of ATP by an increase in the fraction of protonated Asp-396 in the dark (32,56). Simulations suggest that Asp-396 then fully adopts the conformation in which it is hydrogenbonded to the backbone of Met-381 (Leu-378 in CPH1) and allows for an efficient electron transfer pathway from Trp-400 to the flavin (56).…”

“…It has been observed that the presence of ATP stabilizes the flavin neutral radical in the plant cryptochrome (39,44) and increases its yield (32). Therefore, the influence of ATP on the photoreaction of the mutant was investigated.…”

“…Effect of ATP on the Photoreaction of the Mutant-ATP binding leads to an increased yield (32) and stability (39,44) of the radical photoproduct. It is likely that this effect is also of physiological significance because of the high binding affinity determined in vitro (10).…”

“…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). In plant cryptochromes, proton transfer is required for the antagonizing effect of green light found in vivo (18,19,33) because only the protonated radical absorbs green light to a significant extent.…”