Determination of the binding sites between cytochrome f and plastocyanin
Abstract
Cytochrome f and plastocyanin are photosynthetic electron transport catalysts found in chloroplasts. Experiments have shown positively charged residues of cytochrome f and negatively charged residues of plastocyanin to be involved with the enzymatic transfer of electrons from cytochrome f to plastocyanin. Plastocyanin has two regions which are highly negatively charged, residues 42-45 (DEDE) and 59-61 (EED). Specific chemical modifications within either of these regions on plastocyanin greatly inhibits its reduction by cytochrome f. In the work reported here, turnip cytochrome f and spinach plastocyanin have been covalently linked using the water-soluble carbodiimide, EDC. The stoichiometry of the cytochrome f: plastocyanin adduct was 1:1 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectral characterization, and automated sequence analysis. The cytochrome f: plastocyanin adduct was analyzed to determine the binding sites between the two catalysts. Cyanogen bromide, trypsin, and endoproteinase Asp-N cleavage products were purified by reverse phase high performance liquid chromatography. Selected fragments were analyzed by automated sequencing. Aspartate-44 and glutamates-59 and 60 of plastocyanin were determined to be covalently linked to cytochrome f. Aspartate-44 was linked to lysine-187 of cytochrome f. The sites of linkage to cytochrome f for glutamates-59 and 60 were not identified. This result identifies the negatively charged region of plastocyanin, residues 42-45, as a binding site for cytochrome f and is the first identification of its complementary binding sites on cytochrome f as the positively charged region, residues 180-187 (KQIIRKEK). Also, the cytochrome f: plastocyanin adduct was tested for its ability to donate electrons to photosystem I. Photosystem I activity was assayed by plastocyanin-dependent methyl-viologen reduction. Neither the cytochrome f: plastocyanin adduct nor cytochrome f would support electron transfer through photosystem I; however, the addition of free plastocyanin restored full photosystem I activity. This suggests that plastocyanin has the same binding domain for cytochrome f and photosystem I and the same donor-acceptor site of electron transfer.
Degree
Ph.D.
Advisors
Krogmann, Purdue University.
Subject Area
Biochemistry
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