The redox state of cytochrome b559 in spinach chloroplasts

Abstract

Spinach chloroplasts suspended in a mixture of redox buffers were titrated under anaerobic conditions by adding small amounts of oxidant (potassium ferricyanide) or reductant (reduced methyl viologen). At specific redox potentials, measured with a platinum electrode, samples of chloroplasts were removed, frozen to −196 °;C, and single-beam absorption spectra were measured before and after actinic irradiation at −196 °C. The extent of the low temperature light-induced reduction of C-550 and oxidation of cytochrome b 559 was determined as a function of redox potential. Difference spectra between samples at different redox potentials were used to determine titration curves for the chemically induced changes. With intact chloroplasts at 0 °C the midpoint potential of cytochrome b 559 was found to be + 450 mV. Titration of Tris-washed chloroplasts at 20 °C showed that cytochrome b 559 is oxidized at lower potentials and over a wider range of potentials than in intact chloroplasts. A cytochrome b 559 with a still lower midpoint potential (dithionite reducible but not ascorbate reducible) was also demonstrated in intact chloroplasts which had not been subjected to treatments which modify the normal high potential cytochrome b 559.