Time-Resolved Infrared Spectroscopy on Plant Cryptochrome - Relevance of Proton Transfer and ATP Binding for Signaling.

Plant cryptochromes are light receptors in land plants and algae with very diverse functions such as circadian timing and lifecycle progression. The receptor consists of a photolyase homology region (PHR) binding the flavin chromophore and a C-terminal extension (CCT) responsible for signaling. The reputed signaling state, the flavin neutral radical, is formed by a femtosecond electron transfer and microsecond proton transfer to the excited, oxidized flavin. Subsequently, a 500 mu s loss of beta-sheet structure , similar to 25 angstrom away from flavin was resolved and suggested to be part of the signal conduction to the CCT. Here, we performed time-resolved, stepscan Fourier transform IR spectroscopy on the PHR of the plant cryptochrome pCRY (formerly CPH1) from Chlamydomonas reinhardtii. In a mutant lacking the proton donor aspartic acid 396 only the flavin anion radical is formed, but we observed the loss of beta-sheet structure with a time constant of 1.3 ms, similar to the 500 mu s of the wild type. This finding implies that the anion radical may be considered signaling-competent. In the steady state, a variation of external pH up to 8.3 did not have any effect on the difference spectra including the protonated state of Asp396. However, we detected the prominent loss of beta-sheet structure by illumination only in the presence of adenosine triphosphate (ATP). We conclude that the bound ATP stabilizes these light-induced changes in secondary structure to ensure a physiological lifetime compatible with signaling by plant cryptochrome.