Light-induced absorbance changes at −170 °C with spinach chloroplasts: Charge separation and field effect

Abstract

Absorbance changes induced by illumination of spinach chloroplasts at −170° were recorded either with a single-beam kinetic spectrophotometer or with a double-beam differential spectrophotometer, in the range 500–560 nm. 1. 1. The difference spectrum of absorbance changes measured with the kinetic apparatus indicates the reduction of C-550 (peaks at 542, 547.5 nm) and the oxidation of cytochrome b 559 (556 nm). There is an additional peak at 518 nm. The kinetics are nearly identical at 542 and 518 nm, and about twice slower at 556 nm. 2. 2. It is shown that C-550, cytochrome b 559 and the 518-nm effect all belong to Photosystem II. The corresponding absorbance changes do not occur upon illumination at −170° of chloroplasts that received one flash in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea and of hydroxylamine before being cooled. The kinetics under illumination by far-red light confirm the attribution to Photosystem II. Another type of absorbance change was identified, with a smooth difference spectrum peaking at 530 nm; it is attributed to Photosystem I. 3. 3. In the presence of an increasing concentration of ferricyanide, a progressive and parallel decrease of the magnitude of absorbance changes is observed at 518 and 556 nm. Ferricyanide has little effect on absorbance changes at 542 nm. 4. 4. Difference spectra (between a cuvette illuminated at −170° and a reference cuvette) recorded with the double-beam spectrophotometer are nearly identical with the Photosystem II part of the absorbance changes. The peak at 518 nm appears to be stable for at least 10 min. In the presence of ferricyanide a parallel decrease of the peaks at 518 and 556 nm is observed. We propose that the 518 nm effect observed at −170° is due to a local field effect, that needs the reduction of the primary electron acceptor, and that is influenced by the redox state of the secondary electron donors of Photosystem II. A possible similar effect occurring in Photosystem I would be of much smaller amplitude.