Photosynthetic Reactions by Lysed Protoplasts and Particle Preparations from the Blue-Green Alga, Phormidium luridum

Reactions of photosynthetic electron transport and photophosphorylation were studied in preparations from the blue-green alga, Phormidium luridum. Osmotic lysis of protoplasts proved to be a superior technique for the production of cell-free preparations with high enzymatic activity. Such lysed protoplasts sustain high rates of photophosphorylation coupled to the photo-reduction of NADP⁺ or ferricyanide. P/2e⁻ ratios close to unity were routinely observed. The same preparations, and also those prepared by grinding the cells in solutions containing sucrose or ethylene glycol, are active in cyclic photophosphorylation mediated by phenazine methosulfate or dichloro-phenolindophenol. The particles prepared by grinding the cells are, however, inactive in non-cyclic photophosphorylation.

Extensive washing of the membranes with solutions containing sucrose removes the majority of the residual soluble fraction of the algal cell which includes cytochromes C₅₅₄ and C₅₄₉ and phycocyanin. Cyclic photophosphorylation activity is unimpaired by this treatment, but is abolished when the membranes are washed with very dilute buffers. This activity is restored by the addition of a soluble protein which is not a known redox constituent such as cytochrome C₅₅₄ or plastocyanin, and may be a coupling factor.

Analysis of the well-washed membranes by low temperature (77°K) difference spectrophotometry reveals the presence of cytochrome b₆ and a bound form of cytochrome C₅₅₄ in proportions similar to that found in higher plant chloroplasts. The concentration of the membrane-bound cytochrome C₅₅₄, relative to cytochrome b₆ is not altered by extensive washing, sonication or treatment with 1% digitonin. This indicates that this cytochrome is an integral component of the cytoplasmic lamellae and we suggest that it is of functional significance. The soluble form of cytochrome C₅₅₄, which is present in concentrations about 3-fold higher than the bound form, depending upon growth conditions, is not essential for cyclic photophosphorylation. The concentration of cytochrome b₆: chlorophyll a was found to be 1:500.

Under the conditions employed, we were unable to detect a bound form of the low potential cytochrome C₅₄₉.