Isolation of a Cytochrome b6-f Particle from Chloroplasts

Abstract

System 1 particles were prepared from lettuce chloroplasts by the addition of digitonin and differential centrifugation as described previously (Nelson, N., Dreschler, Z., and Neumann, J. (1970) J. Biol. Chem. 245, 143), but higher detergent concentrations were used. A cytochrome b6-f particle was isolated from System 1 particles by the addition of protamine sulfate and centrifugation. The particle was purified by Bio-Gel and DEAE-column chromatography and ammonium sulfate precipitation. The presence of detergents during purification was required in order to prevent aggregation of the cytochromes. The ratio of cytochrome b6 to cytochrome f in the purified particle was close to 2. The particle contained non-heme iron and phospholipids. It was devoid of chlorophyll but contained carotenes. The calculated minimum molecular weight of the particle was 103,000 per 1 heme c. The molar extinction coefficient of the cytochromes in the particle was determined by measuring the absorbance of the pyridine hemochrome complexes. For cytochrome b6 the molar extinction coefficients obtained were as follows: reduced minus oxidized at 563 nm minus 575 nm, 24,400; and at 563 nm minus 542 nm, 20,700. For cytochrome f the molar extinction coefficients obtained were as follows: reduced minus oxidized at 554 nm minus 538 nm, 20,600; and at 554 nm minus 565 nm 23,000. The E0 of cytochrome f in the particle is +330 mvolts and that of cytochrome b6 is -100 mvolts. The cytochrome b6-f particle from chloroplasts shows similarities with the cytochrome b-c1 complex (Complex III) from mitochondria. Some implications of the present findings for the operation of photosynthetic electron transport pathways are discussed.