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Wenz, T., Hellwig, P., Macmillan, F. 
ORCID: https://orcid.org/0000-0002-2410-4790, Meunier, B. and Hunte, C.
(2006)
Probing the role of E272 in quinol oxidation of mitochondrial complex III.
Biochemistry, 45 (30).
pp. 9042-9052.
ISSN 0006-2960
Abstract
Bifurcated electron transfer during ubiquinol oxidation is the key reaction of complex III catalysis, but the molecular basis of this process is still not clear. E272 of the conserved cytochrome b PEWY motif has been suggested as a ligand and proton acceptor for ubiquinol oxidation at center P. We introduced the two replacement mutations, E272D and E272Q, into the mitochondrially encoded cytochrome b gene by biolistic transformation to study their effects on substrate binding and catalysis. Both substitutions resulted in a lower ubiquinol cytochrome c reductase activity and affect the KM for ubiquinol. The E272 carboxylate stabilizes stigmatellin binding, and in accordance, both variants are resistant to stigmatellin. Large structural changes in the cofactor environment as well as in the binding pocket can be excluded. The mutations do not perturb the midpoint potentials of the heme groups. The sensitivity toward the respective distal and proximal niche inhibitors HDBT and myxothiazol is retained. However, both mutations provoke subtle structural alterations detected by redox FTIR. They affect binding and oxidation of ubiquinol, and they promote electron short-circuit reactions resulting in production of reactive oxygen species. The aspartate substitution modifies the environment of the reduced Rieske protein as monitored by EPR. Both variants alter the pH dependence of the enzyme activity. Diminished activity at low pH coincides with the loss of one protonatable group with a pK(a) of similar to 6.2 compared to three pK(a) values in the wild type, supporting the role of E272 in proton transfer. The conserved glutamate appears to influence the accurate formation of the enzyme-substrate complex and to govern the efficiency of catalysis.
| Item Type: | Article |
|---|---|
| Faculty \ School: | Faculty of Science > School of Chemistry (former - to 2024) |
| UEA Research Groups: | Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017) Faculty of Science > Research Groups > Chemistry of Life Processes Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Chemistry of Light and Energy |
| Depositing User: | Rachel Smith |
| Date Deposited: | 18 Jul 2011 12:15 |
| Last Modified: | 24 Sep 2024 09:53 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/33899 |
| DOI: | 10.1021/bi060280g |
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