The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Abstract

bCytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ϳ30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ⌬cydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.

Cytochrome bd from Escherichia coli catalyzes peroxynitrite decomposition

“…This confirms that in the isolated enzyme all the three hemes, including the di-heme active site components, heme b 595 and heme d, were present as expected for a bd-type oxidase [14]. Based on recent reports [23][24][25], this suggests that the purified enzyme likely included the newly discovered small subunit CydX. The enzyme concentration was determined from the dithionitereduced-minus-'as prepared' difference absorption spectrum using Δε 628-607 = 10.8 mM −1 cm −1 [30].…”

“…Cytochrome bd is composed of two major membrane-spanning polypeptides, subunits I (CydA, 57 kDa) and II (CydB, 43 kDa), and a small protein, named CydX (4 kDa). The latter has been recently shown to be essential for enzymatic activity and proposed to be an additional subunit of the complex, needed for either the assembly or the stability of the enzyme [23][24][25]. Cytochrome bd bears two b-type hemes, the low-spin b 558 and the high-spin b 595 , and one chlorin, heme d. Heme b 558 accepts electrons from the quinol substrate, whereas heme d is the site where the oxygen chemistry takes place.…”

The Terminal Oxidase cbb 3 Functions in Redox Control of Magnetite Biomineralization in Magnetospirillum gryphiswaldense

“…This might be caused by the low efficiency of bd oxidase, which is not able to pump protons but accepts only electrons directly from the quinol pool for O 2 reduction, while cytochrome c oxidase cbb 3 is more efficient at creating the charge gradient for ATP synthesis via the bc-c-cbb 3 branch (22). An even stronger delay of growth was observed for the ⌬cbb 3 and ⌬aa 3 ⌬cbb 3 mutants in microaerobic nitrate medium.…”

“…The bd-type quinol oxidases provide an alternative branch and accept electrons directly from the quinol pool for O 2 reduction (18). Although bd quinol oxidases do not pump protons and therefore are less efficient at creating the charge gradient for ATP synthesis than HCOs, they have been found to have a higher affinity for O 2 than other cytochrome oxidases (22) and therefore were proposed to function under low-O 2 conditions (23). However, their physiological function has remained unclear.…”

The CydDC Family of Transporters and Their Roles in Oxidase Assembly and Homeostasis

“…All known members of the bd-family use quinol as a substrate, receiving electrons commonly from either ubiquinol or menaquinol. With a three-dimensional structure yet to be elucidated, existing data show that cytochrome bd-I is a trimer of three membrane polypeptides, subunits I (CydA) and II (CydB) (Kita, Konishi, & Anraku, 1984) and CydX (Van Orsdel et al, 2013). Three haems are associated with the oxidase in a 1:1:1 stoichiometry peroxidase complex.…”