Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo 13C-Labeled Methyl and Methoxy Substituents Full article
| Journal |
Journal of Biological Chemistry
ISSN: 0021-9258 , E-ISSN: 1083-351X |
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| Output data | Year: 2011, Volume: 286, Number: 12, Pages: 10105-10114 Pages count : 10 DOI: 10.1074/jbc.M110.206821 | ||||||||
| Tags | Amino acids; Catalyst activity; Cell membranes; Electronic structure; Escherichia coli; Functional groups; Proteins | ||||||||
| Authors |
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| Affiliations |
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Funding (4)
| 1 | National Institutes of Health | GM 62954 |
| 2 | National Institutes of Health | S10-RR15878 |
| 3 | United States Department of Energy | DE-FG02-08ER15960 |
| 4 | United States Department of Energy | DE-FG02-87ER13716 |
Abstract:
The cytochromebo3ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduc-tion of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo3 as well as the D75E and D75H mutant proteins were pre-pared with ubiquinone-8 13C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence ofL-me-thionine with the side chain methyl group13C-labeled. The13C-labeled quinone isolated from cytochromebo3was also used for the generation of model anion radicals in alcohol. Two-dimen-sionalpulsedEPRandENDORwereusedforthestudyofthe13C methyl and methoxy hyperfine couplings in the semiquinone generatedinthethreeproteinsindicatedaboveandinthemodel system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity.
Cite:
Lin M.T.
, Shubin A.A.
, Samoilova R.I.
, Narasimhulu K.V.
, Baldansuren A.
, Gennis R.B.
, Dikanov S.A.
Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo 13C-Labeled Methyl and Methoxy Substituents
Journal of Biological Chemistry. 2011.
V.286. N12. P.10105-10114. DOI: 10.1074/jbc.M110.206821
WOS
Scopus
РИНЦ
ANCAN
OpenAlex
Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo 13C-Labeled Methyl and Methoxy Substituents
Journal of Biological Chemistry. 2011.
V.286. N12. P.10105-10114. DOI: 10.1074/jbc.M110.206821
WOS
Scopus
РИНЦ
ANCAN
OpenAlex
Files:
Full text from publisher
Dates:
| Submitted: | Nov 25, 2010 |
| Accepted: | Jan 13, 2011 |
| Published online: | Jan 19, 2011 |
| Published print: | Mar 25, 2011 |
Identifiers:
| Web of science: | WOS:000288547000025 |
| Scopus: | 2-s2.0-79953195121 |
| Elibrary: | 16983874 |
| Chemical Abstracts: | 2011:348865 |
| Chemical Abstracts (print): | 154:479082 |
| OpenAlex: | W1972996979 |