Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for assembly and stability of the di‐heme active site

Abstract

Edited by Miguel De la Rosa Keywords:Ubiquinol cytochrome bd oxidase CydAB CydX UV/vis difference spectroscopy Escherichia coli a b s t r a c t Cytochrome bd ubiquinol oxidase uses the electron transport from ubiquinol to oxygen to establish a proton gradient across the membrane. The enzyme complex consists of subunits CydA and B and contains two b-and one d-type hemes as cofactors. Recently, it was proposed that a third subunit named CydX is essential for the function of the complex. Here, we show that CydX is indeed a subunit of purified Escherichia coli cytochrome bd oxidase and that the small protein is needed either for the assembly or the stability of the active site di-heme center and, thus, is essential for oxidase activity. Structured summary of protein interactions:cydA physically interacts with cydB by affinity technology (View interaction) cydA physically interacts with cydB by molecular sieving (View interaction) cydB, cydA and cydX physically interact by molecular sieving (View interaction) cydB, cydA, and cydX physically interacts by affinity technology (1, 2)

Cytochrome bd Displays Significant Quinol Peroxidase Activity

“…However, recent mutational studies in E. coli, Brucella abortus and Shewanella oneidensis suggested that the small protein, CydX (37, 64 and 38 amino acids in E. coli, B. abortus and S. oneidensis , resp.) is important for assembly, stability and activity of cytochrome bd in vivo and in vitro 63747576. The presence of CydX has also been confirmed in purified cytochrome bd 75.…”

“…is important for assembly, stability and activity of cytochrome bd in vivo and in vitro 63747576. The presence of CydX has also been confirmed in purified cytochrome bd 75. To confirm the presence of CydX in our preparation we performed a mass-spectrometric analysis.…”

Crosstalk between sugarcane and a plant-growth promoting Burkholderia species

“…These genes are highly induced in the sugarcane rhizosphere (Supplementary Table S5), cydA 34-fold, cydB 27-fold, cbdA 112-fold, cbdB 49-fold. CydX , a protein essential for assembly and stability of the cytochrome complex43, is upregulated 21-fold. Expression of cytochrome bo is, however, unchanged.…”

Synthesis and Biological Screening of New Lawson Derivatives as Selective Substrate‐Based Inhibitors of Cytochrome bo3 Ubiquinol Oxidase from Escherichia coli

“…Production of cytochrome bd-I from E. coli: Production of cytochrome bd-I oxidase from E. coli in E. coli C43(DE3) Δbo 3 cells transformed with the pet17b-cydABX-StrepII plasmid. [46] 1 mL of 50 % Glycerol stock was added to 50 mL LB-Carb-Kan (50 μg/mL carbenicillin; 50 mg/mL kanamycin) and incubated at 175 rpm at 37°C for 8 h. Preculture was transferred to 1 L LB-Carb-Kan to grow over night. Production culture of 2.5 L LB-Kan was inoculated with 70 mL of overnight culture supplemented with 0.025 mM IPTG to start basal production from the beginning.…”