DOI: 10.1073/pnas.94.19.10128. Glutamic acid 286 in subunit I of cytochrome <i>bo</i> <sub>3</sub> is involved in proton translocation

Glutamic acid 286 in subunit I of cytochrome bo 3 is involved in proton translocation

10.1073/pnas.94.19.10128

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Glutamic acid 286 (E286; Escherichia coli cytochrome bo 3 numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome bo 3 vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O 2 . We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.

Bibliography

Verkhovskaya, M. L., GarcÃ¬a-Horsman, A., Puustinen, A., Rigaud, J.-L., Morgan, J. E., Verkhovsky, M. I., & WikstrÃ¶m, M. (1997). Glutamic acid 286 in subunit I of cytochrome bo 3 is involved in protonâtranslocation. Proceedings of the National Academy of Sciences, 94(19), 10128â10131.

Authors 7

Marina L. Verkhovskaya (first)
Arturo Garcìa-Horsman (additional)
Anne Puustinen (additional)
Jean-Louis Rigaud (additional)
Joel E. Morgan (additional)
Michael I. Verkhovsky (additional)
Mårten Wikström (additional)

References 28 Referenced 108

10.1038/356301a0
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M Wikström, J E Morgan, G Hummer, W H Woodruff, M I Verkhovsky Frontiers of Cellular Bioenergetics, eds S Papa, F Guerrieri, J M Tager (Plenum, New York, , in press. (1997). / Frontiers of Cellular Bioenergetics by Wikström M (1997)
10.1007/BF00762854
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Puustinen A. Bailey J. A. Dyer R. B. Mecklenburg S. L. Wikström M. & Woodruff W. H. (1997) Biochemistry in press.
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Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:31 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 3:22 p.m.)
Indexed	1 month, 4 weeks ago (June 27, 2025, 6:35 a.m.)
Issued	27 years, 11 months ago (Sept. 16, 1997)
Published	27 years, 11 months ago (Sept. 16, 1997)
Published Online	27 years, 11 months ago (Sept. 16, 1997)
Published Print	27 years, 11 months ago (Sept. 16, 1997)

Funders 0

None

BibTeX

@article{Verkhovskaya_1997, title={Glutamic acid 286 in subunit I of cytochrome bo 3 is involved in proton translocation}, volume={94}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.94.19.10128}, DOI={10.1073/pnas.94.19.10128}, number={19}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Verkhovskaya, Marina L. and Garcìa-Horsman, Arturo and Puustinen, Anne and Rigaud, Jean-Louis and Morgan, Joel E. and Verkhovsky, Michael I. and Wikström, Mårten}, year={1997}, month=sep, pages={10128–10131} }

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  "abstract": "<jats:p>\n            Glutamic acid 286 (E286;\n            <jats:italic>Escherichia coli</jats:italic>\n            cytochrome\n            <jats:italic>bo</jats:italic>\n            <jats:sub>3</jats:sub>\n            numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome\n            <jats:italic>bo</jats:italic>\n            <jats:sub>3</jats:sub>\n            vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O\n            <jats:sub>2</jats:sub>\n            . We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.\n          </jats:p>",
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