The carboxy-terminal insert in the Q-loop is needed for functionality of Escherichia coli cytochrome bd-I.
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 Ile318-Met322, Gln338-Asp342, Tyr353-Leu357, and Thr368-Ile372 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.
['Goojani|Hojjat Ghasemi|HG|', 'Konings|Julia|J|', 'Hakvoort|Henk|H|', 'Hong|Sangjin|S|', 'Gennis|Robert B|RB|', 'Sakamoto|Junshi|J|', 'Lill|Holger|H|', 'Bald|Dirk|D|']
D000409:Alanine D000595:Amino Acid Sequence D002462:Cell Membrane D003573:Cytochrome b Group D045222:Electron Transport Chain Complex Proteins D004926:Escherichia coli D029968:Escherichia coli Proteins D006418:Heme D016296:Mutagenesis D050505:Mutant Proteins D010088:Oxidoreductases D010101:Oxygen Consumption D017433:Protein Structure, Secondary D013329:Structure-Activity Relationship
Q-loop Cytochrome bd Biochemistry Mutagenesis Terminal oxidase