Studies on Algal Cytochromes VI: Some Properties and Amino Acid Sequence of Cytochrome c₆ from a Green Alga, Bryopsis maxima

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A photosynthetic c-type cytochrome, cytochrome c₆, was extracted from a green alga, Bryopsis maxima, by cutting and immersing the frozen thalli in phosphate buffer, pH 7.0, and purified by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography and Bio-Gel P-10 gel filtration. The ferrocytochrome c₆ has absorption maxima at 553.5 (α), 523 (β), 417 (γ), 318 (δ), and 275 nm, and the ferricytochrome at 695, 528, and 411 (γ). The molecular weight was estimated to be about 10, 000 from Sephadex G-75 gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The midpoint redox potential for the cytochrome was determined by equilibrium titration with a ferroand ferricyanide system to be 0.385 volt at pH 7.0. Isoelectric points for ferroand ferricytochromes were determined by density gradient isoelectric focusing electrophoresis to be at pH 3.91 and 4.02, respectively. The complete amino acid sequence of the cytochrome was determined by Edman degradation and by carboxypeptidase digestions of the Cm-cytochrome, 6 staphylococcal protease peptides and 5 lysyl endopeptidase peptides. The cytochrome contained 88 amino acid residues, giving a molecular weight of 9, 904 including 1 mol of heme c. The sequence is as follows: GGDLEIGADVFTGNCAACHAGGANSVEPLKTLNKED-V T KY LD G G LSIEAITSQVRNGKGA MPAWSDRLDDEEIDGVVAYVFKNINE-GW. A phylogenetic tree of 13 algal cytochromes c₆ was constructed by comparing the amino acid differences.