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

Proc Natl Acad Sci U S A

Photobioenergetics Research Group, Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia.

Published: December 2004

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 approximately 10 A 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 approximately 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).

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC539716PMC
http://dx.doi.org/10.1073/pnas.0406192101DOI Listing

Publication Analysis

Top Keywords

electron transfer
12
protein engineering
8
cytochrome b562
8
light-induced electron
8
hydrophobic interior
8
protein
6
quinone
5
bound
5
engineering cytochrome
4
b562 quinone
4

Similar Publications

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!