Protein engineering of cytochrome b562 for quinone binding and light-induced electron transfer
dc.contributor.author | Hay, Sam | |
dc.contributor.author | Wallace, Brett | |
dc.contributor.author | Smith, Trevor A | |
dc.contributor.author | Ghiggino, Kenneth | |
dc.contributor.author | Wydrzynski, Thomas | |
dc.date.accessioned | 2015-12-13T22:43:52Z | |
dc.date.available | 2015-12-13T22:43:52Z | |
dc.date.issued | 2004 | |
dc.date.updated | 2015-12-11T10:14:35Z | |
dc.description.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. | |
dc.identifier.issn | 0027-8424 | |
dc.identifier.uri | http://hdl.handle.net/1885/79392 | |
dc.publisher | National Academy of Sciences (USA) | |
dc.source | PNAS - Proceedings of the National Academy of Sciences of the United States of America | |
dc.subject | 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 | |
dc.title | Protein engineering of cytochrome b562 for quinone binding and light-induced electron transfer | |
dc.type | Journal article | |
local.bibliographicCitation.issue | 51 | |
local.bibliographicCitation.lastpage | 17680 | |
local.bibliographicCitation.startpage | 17675 | |
local.contributor.affiliation | Hay, Sam, College of Medicine, Biology and Environment, ANU | |
local.contributor.affiliation | Wallace, Brett, College of Medicine, Biology and Environment, ANU | |
local.contributor.affiliation | Smith, Trevor A, University of Melbourne | |
local.contributor.affiliation | Ghiggino, Kenneth, University of Melbourne | |
local.contributor.affiliation | Wydrzynski, Thomas, College of Medicine, Biology and Environment, ANU | |
local.contributor.authoremail | u9114707@anu.edu.au | |
local.contributor.authoruid | Hay, Sam, u4022950 | |
local.contributor.authoruid | Wallace, Brett, u4029765 | |
local.contributor.authoruid | Wydrzynski, Thomas, u9114707 | |
local.description.notes | Imported from ARIES | |
local.description.refereed | Yes | |
local.identifier.absfor | 100104 - Genetically Modified Animals | |
local.identifier.ariespublication | MigratedxPub7841 | |
local.identifier.citationvolume | 101 | |
local.identifier.doi | 10.1073/pnas.0406192101 | |
local.identifier.scopusID | 2-s2.0-11144242802 | |
local.identifier.uidSubmittedBy | Migrated | |
local.type.status | Published Version |