Protein engineering of cytochrome b562 for quinone binding and light-induced electron transfer
Date
2004
Authors
Hay, Sam
Wallace, Brett
Smith, Trevor A
Ghiggino, Kenneth
Wydrzynski, Thomas
Journal Title
Journal ISSN
Volume Title
Publisher
National Academy of Sciences (USA)
Abstract
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.
Description
Keywords
Keywords: 1,4 benzoquinone; 2,3 dimethoxy 5 methyl 1,4 benzoquinone; chlorine; chlorophyll; cysteine; cytochrome b; cytochrome b562; hydroquinone; porphyrin; quinone derivative; sulfur; unclassified drug; zinc; addition reaction; article; binding site; chemical rea Artificial photosynthesis; Chlorophyll analog; Cysteine; Photosynthetic reaction center; Zinc chlorin
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Source
PNAS - Proceedings of the National Academy of Sciences of the United States of America
Type
Journal article