The carboxy-terminal insert in the Q-loop is needed for functionality of Escherichia coli cytochrome bd-I.

Autor: Goojani HG; Department of Molecular Cell Biology, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, the Netherlands., Konings J; Department of Molecular Cell Biology, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, the Netherlands., Hakvoort H; Department of Molecular Cell Biology, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, the Netherlands., Hong S; Department of Biochemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801, United States., Gennis RB; Department of Biochemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801, United States., Sakamoto J; Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Kawazu 680-4, Iizuka, Fukuoka-ken 820-8502, Japan., Lill H; Department of Molecular Cell Biology, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, the Netherlands., Bald D; Department of Molecular Cell Biology, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, the Netherlands. Electronic address: d.bald@vu.nl.
Jazyk: angličtina
Zdroj: Biochimica et biophysica acta. Bioenergetics [Biochim Biophys Acta Bioenerg] 2020 Jun 01; Vol. 1861 (5-6), pp. 148175. Date of Electronic Publication: 2020 Feb 12.
DOI: 10.1016/j.bbabio.2020.148175
Abstrakt: Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile 318 -Met 322 , Gln 338 -Asp 342 , Tyr 353 -Leu 357 , and Thr 368 -Ile 372 as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2020. Published by Elsevier B.V.)
Databáze: MEDLINE
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