Figure 3.

Identification of the intermediate and final product of PChlide photoreduction reaction. (A) Fluorescence excitation spectra (top lines) and second derivatives (bottom lines) of the initial PChlide (DA,F675 and DA,F685), the intermediate F685, formed after illu mination of the sample with white light (33 mE⋅m⁻²⋅s⁻¹) for 10 min at −50°C (WL10′ at −50C, F685), and the Chlide F675, formed after illumination of the sample with a light flash given at −50°C and subsequent heating of the sample in the dark to −25°C (1×F at50C+DA-25C). All fluorescence spectra were recorded at −196°C. Note that the formation of the intermediate F682 does not lead to degeneration of B_x and B_y bands in the excitation spectrum whereas formation of the final product (F675) does. (B) HPLC identification of Chlide formed after a single light flash at −50°C and subsequent heating to −25°C (1×F at-50C+DA5′ at−25C) and after illumination with continuous white light at +22°C for 10 min (WL10′ at +22°C). The pigments were extracted from the frozen samples with acetone in complete darkness. The arrows indicate retention times of pure PChlide and Chlide. The pigments were detected in the column eluant fluorometrically under excitation at 435 and emission at 675 nm. (C) EPR signal that appears after illumination of the reaction mix with white light for 10 min at −50°C (WL10′ at−50C). No such signal was detected when sample was heated to −50°C and kept at this temperature for 10 min without illumination (DA10′ at −50C). For EPR spectral recording, the frozen samples were cooled to −170°C and assayed at a frequency of 9.463 GHz and microwave power = 10 mW.