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

Hay, Sam; Wallace, Brett; Smith, Trevor A; Ghiggino, Kenneth; Wydrzynski, Thomas

Description

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...[Show more]

dc.contributor.authorHay, Sam
dc.contributor.authorWallace, Brett
dc.contributor.authorSmith, Trevor A
dc.contributor.authorGhiggino, Kenneth
dc.contributor.authorWydrzynski, Thomas
dc.date.accessioned2015-12-13T22:43:52Z
dc.date.available2015-12-13T22:43:52Z
dc.identifier.issn0027-8424
dc.identifier.urihttp://hdl.handle.net/1885/79392
dc.description.abstractThe 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.publisherNational Academy of Sciences (USA)
dc.sourcePNAS - Proceedings of the National Academy of Sciences of the United States of America
dc.subjectKeywords: 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.titleProtein engineering of cytochrome b562 for quinone binding and light-induced electron transfer
dc.typeJournal article
local.description.notesImported from ARIES
local.description.refereedYes
local.identifier.citationvolume101
dc.date.issued2004
local.identifier.absfor100104 - Genetically Modified Animals
local.identifier.ariespublicationMigratedxPub7841
local.type.statusPublished Version
local.contributor.affiliationHay, Sam, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationWallace, Brett, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationSmith, Trevor A, University of Melbourne
local.contributor.affiliationGhiggino, Kenneth, University of Melbourne
local.contributor.affiliationWydrzynski, Thomas, College of Medicine, Biology and Environment, ANU
local.bibliographicCitation.issue51
local.bibliographicCitation.startpage17675
local.bibliographicCitation.lastpage17680
local.identifier.doi10.1073/pnas.0406192101
dc.date.updated2015-12-11T10:14:35Z
local.identifier.scopusID2-s2.0-11144242802
CollectionsANU Research Publications

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