Protein engineering of cytochrome b562 for quinone binding and light-induced electron transfer.
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).
Hay
Sam
S
Photobioenergetics Research Group, Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia. hay@rsbs.anu.edu.au
Wallace
Brett B
BB
Smith
Trevor A
TA
Ghiggino
Kenneth P
KP
Wydrzynski
Tom
T
D001665:Binding Sites; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004579:Electron Transport / Q000528:radiation effects*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005453:Fluorescence; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D008027:Light; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011166:Porphyrins / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011809:Quinones / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis; D017186:Titrimetry
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D011166:Porphyrins; D011809:Quinones; D007532:Isoleucine; C017850:cytochrome b562, E coli