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.

中文翻译：

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Q-环中的羧基末端插入物是大肠杆菌细胞色素bd-I的功能所必需的。

细胞色素bd是原核呼吸链的一个组成部分，在生理压力和致病性过程中很重要。来自喹诺酚底物的电子通过CydA亚基中的血红素基团传递，并用于还原分子氧。CydA靠近喹诺酮结合位点，显示出称为Q环的周质亲水环，这对喹诺氧化必不可少。在该环的羧基末端部分，来自大肠杆菌和其他变形细菌的CydA带有约60个功能未知的残基插入物。在当前的工作中，我们证明了通过大肠杆菌细胞色素bd-I的转化可以提高多重删除菌株大肠杆菌MB43ΔcydA（ΔcydAΔcydBΔappBΔcyoBΔnuoB）的生长，并且我们可以利用这一点评估Q环突变体的系统。删除细胞色素bd-I Q环插入片段可消除MB43∆cydA的生长恢复。缺少插入片段的将热色素土细菌或结核分枝杆菌CydA的Q环替换为细胞色素bd-I Q环并没有增强MB43ΔcydA的生长，而从大肠杆菌的细胞色素bd换为Q环。 -II恢复增长。丙氨酸扫描实验确定了细胞色素bd-I Q环插入区域Ile318-Met322，Gln338-Asp342，Tyr353-Leu357和Thr368-Ile372对于酶功能很重要。那些完全无法恢复MB43ΔcydA生长的突变体也缺乏耗氧活性和血红素吸收峰。此外，我们无法从这些无活性的突变体中分离出细胞色素bd-I。