Protein engineering of cytochrome b562 for quinone binding and light-induced electron transfer

Sam Hay, Brett B. Wallace, Trevor A. Smith, Kenneth P. Ghiggino, Tom Wydrzynski

    Research output: Contribution to journalArticlepeer-review


    The central photochemical reaction in photosystem II of green algae and plants and the reaction center of some photosynthetic bacteria involves a one-electron transfer from a light-activated chlorin complex to a bound quinone molecule. Through protein engineering, we have been able to modify a protein to mimic this reaction. A unique quinone-binding site was engineered into the Escherichia coli cytochrome 6552 by introducing a cysteine within the hydrophobic interior of the protein. Various quinones, such as p-benzoquinone and 2,3-dimethoxy-5-methyl-1,4-benzoquinone, were then covalently attached to the protein through a cysteine sulfur addition reaction to the quinone ring. The cysteine placement was designed to bind the quinone ≈ 10 Å from the edge of the bound porphyrin. Fluorescence measurements confirmed that the bound hydroquinone is incorporated toward the protein's hydrophobic interior and is partially solvent-shielded. The bound quinones remain redox-active and can be oxidized and rereduced in a two-electron process at neutral pH. The semiquinone can be generated at high pH by a one-electron reduction, and the midpoint potential of this can be adjusted by ≈500 mV by binding different quinones to the protein. The heme-binding site of the modified cytochrome was then reconstituted with the chlorophyll analogue zinc chlorin e6. By using EPR and fast optical techniques, we show that, in the various chlorin-protein-quinone complexes, light-induced electron transfer can occur from the chlorin to the bound oxidized quinone but not the hydroquinone, with electron transfer rates in the order of 108 s-1.
    Original languageEnglish
    Pages (from-to)17675-17680
    Number of pages5
    JournalProceedings of the National Academy of Sciences of the United States of America
    Issue number51
    Publication statusPublished - 21 Dec 2004


    • Artificial photosynthesis
    • Chlorophyll analog
    • Cysteine
    • Photosynthetic reaction center
    • Zinc chlorin


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