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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2004-12, Vol.101 (51), p.17675-17680
• Main Authors: Hay, Sam, Wallace, Brett B, Smith, Trevor A, Ghiggino, Kenneth P, Wydrzynski, Tom
• Format: Article
• Language: English
• Subjects: Binding Sites; Biochemistry; Biological Sciences; Cellular biology; Chlorophyll; Cytochrome b Group - chemistry; Cytochrome b Group - genetics; Cytochrome b Group - metabolism; Electron Transport - radiation effects; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fluorescence; Isoleucine - genetics; Isoleucine - metabolism; Light; Models, Molecular; Molecular Structure; Mutation - genetics; Oxidation-Reduction; Photosynthesis; Porphyrins - metabolism; Protein Conformation; Protein Engineering; Proteins; Quinones - chemistry; Quinones - metabolism; Spectrum Analysis; Titrimetry
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0406192101

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 b 562 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 e 6 . 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 10 8 s –1 . photosynthetic reaction center artificial photosynthesis chlorophyll analog zinc chlorin cysteine