Time-Resolved Circular Dichroism of Native Oat Phytochrome Photointermediates

Time-resolved circular dichroism (TRCD) spectral studies of the Pr→ Pfr phototransformation in native oat phytochrome (124 kDa) were performed in the 500-800 nm spectral region from 500 ns to 500 ms after photolysis at 10 °C. The first intermediate observed in our measurements, lumi-R, had an absorption maximum at 695 nm, but its difference TRCD spectrum showed a CD maximum near 660 nm. This result differed from results of previous steady-state CD measurements where the intermediates of the photoconversion were trapped at low temperatures. Possible explanations for the discrepancies in the TRCD results vs low-temperature CD results are discussed. We conclude that the first species trapped in the low-temperature CD experiment is most probably an unrelaxed lumi-R-type intermediate. The subsequent species probed during the Pr → Pfr phototransformation have similar CD spectra. The structural implications of the TRCD results are discussed.