Figure 2.

Steps of PChlide photoreduction resolved by illumination at low temperatures. (A) Fluorescence spectrum of initial sample frozen in the dark to −196°C (INIT. SAMPLE). The same sample was heated in the dark to −50°C, illuminated with a 1-ms flash of white light (1×F), and then cooled to −196°C, where the fluorescence spectrum was recorded. In control experiments, samples were heated in the dark to −25°C, maintained at this temperature for 10 min, and then cooled to −196°C (DA10′). Note that the difference spectrum (LIGHT–DARK, +NADPH) shows that photoactive PChlide has emission maximum at 644 nm, and its photoreduction leads to the formation of an intermediate with fluorescence maximum at 682 nm. No such change was detected in the absence of NADPH (LIGHT–DARK, −NADPH). No changes in PChlide fluorescence around 655 nm were detected under any of the conditions tested. (B) Initial sample was heated in the dark to −50°C, illuminated at this temperature with a 1-ms flash of white light, and then either cooled to −196°C, where its fluorescence was recorded (1×F), or heated in the dark to −25°C, kept at this temperature for 4 min, and then cooled to −196°C (1×F+DA4′). Note that heating of the illuminated sample in the dark without thawing leads to the shift of the emission band of the intermediate product from 682 to 676 nm without any changes in the main PChlide fluorescence band at about 633 nm. All spectra were recorded at −196°C, with an excitation wavelength of 416 nm.