Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi.

The elucidation of the mechanism of photosynthetic electron transport and photosynthetic phosphorylation depends, in part, upon a knowledge of the intermediates in the photosynthetic electron transport chain and the sequence in which they act. Mutant strains of the unicellular green alga Chlamydomonas reinhardi that cannot carry out normal photosynthesis1 can aid in obtaining this knowledge. In this paper we describe results obtained with ac-206, a mutant strain that lacks cytochrome f, and ac-208, a mutant strain that lacks the copper protein, plastocyanin. Since its discovery by Katoh,2 plastocyanin has been presumed to play a role in photosynthetic electron transport. In this paper we present evidence for its participation and site of action with respect to cytochrome f in the photosynthetic electron transport chain of C. reinhardi. Organisms and Methods.-The organisms used in the experiments described below were the wild-type strain of C. reinhardi (137c), and the mutant strains ac-206 and ac-208 derived from the wild type by ultraviolet irradiation.' Cells, in the logarithmic phase of growth, were harvested either from 300-ml shake cultures or from vigorously aerated 12-liter cultures grown in the light at 250C. The growth medium differed from that previously described.4 Tris-acetate buffer, 0.02 M, pH 7.2, was used in place of both phosphate buffer and sodium acetate. Potassium phosphate buffer, 0.001 M, pH 7.0, was also added. Light was provided by daylight fluorescent lamps at an intensity of 4000 lux for cultures of wild type and ac-206, and at 2000 lux for cultures of ac-208. Chloroplast fragments for the measurement of the photoreduction of NADP and DPIP were prepared by the sonic disruption of 5-ml suspensions of cells for 30 sec according to the method described by Levine and Volkmann.5 Chloroplast fragments for the measurement of photosynthetic phosphorylation were prepared by grinding a paste of cells in purified sand. After grinding, the disrupted cells were suspended and washed in the following medium: 0.01 M potassium phosphate buffer, pH 7.5; 0.02M KCl; 0.0025M MgCl2; 0.001 M MgNa2 EDTA; and 0.001 M reduced glutathione. The separation of chloroplast fragments from whole cells was then carried out as described by Levine and Volkmann.5 The photoreduction of NADP and DPIP was measured as previously described.6 Cyclic and noncyclic photosynthetic phosphorylation were measured by the techniques described by Avron7 and Avron and Shavitt.8 Chlorophyll was determinied by a modifications of the procedure of MacKinney.10 Purified PPNR was prepared from wild-type C. reinhardi according to the procedure described by Tagawa and Arnon." Plastocyanin was measured, after partial purification, by a modification of the method of Katoh, Shiratori, and Takamiya.12 Twenty-four liters of cell culture were harvested and cells were resuspended in 0.002 M phosphate buffer, pH 7.0, to a chlorophyll concentration of about 2 mg per ml. All subsequent procedures were carried out in a cold room at 4VC. The cell suspension was added quickly, under rapid stirring, to 4 vol of acetone at about -250C (the temperature after mixing was about -10(C). The cells were then collected as quickly as possible by filtration and resuspended ill 0.01 M phosphate buffer, pH 7.0. The suspension was allowed to stand at 4VC for at least 6 hr, after which the cell debris was centrifuged out at 20,000 X g for 10 min, and the clear supernate was collected. This was then allowed to drain through a column (about 1.3 X 13 cm) of fine-meshed DEAE cellulose (Selectacel DEAE 40) equilibrated with 0.01 Al phosphate buffer, pH 7.0. The column was then eluted with 20 ml of 0.05 M phosphate buffer, pH 7.0, followed by 30 ml of 0.05 M phosphate buffer with 0.15 Al KCl. The column effluejit was collected in 5-ml fractions. The plastocyanin was nearly always confined to the first 20 ml of