Cryo-EM structures of Escherichia coli cytochrome bo3 reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3) have been determined by cryogenic electron microscopy (cryo-EM) in styrene–maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3, and CuB), the redox-active cross-linked histidine–tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I. In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

Cardiolipin enhances the enzymatic activity of cytochrome bd and cytochrome bo3 solubilized in dodecyl-maltoside

Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of mitochondria. CL can increase the activity of integral membrane proteins, in particular components of respiratory pathways. We here report that CL activated detergent-solubilized cytochrome bd, a terminal oxidase from Escherichia coli. CL enhanced the oxygen consumption activity ~ twofold and decreased the apparent KM value for ubiquinol-1 as substrate from 95 µM to 35 µM. Activation by CL was also observed for cytochrome bd from two Gram-positive species, Geobacillus thermodenitrificans and Corynebacterium glutamicum, and for cytochrome bo3 from E. coli. Taken together, CL can enhance the activity of detergent-solubilized cytochrome bd and cytochrome bo3.

Genome Features of Asaia sp. W12 Isolated from the Mosquito Anopheles stephensi Reveal Symbiotic Traits

Asaia bacteria commonly comprise part of the microbiome of many mosquito species in the genera Anopheles and Aedes, including important vectors of infectious agents. Their close association with multiple organs and tissues of their mosquito hosts enhances the potential for paratransgenesis for delivery of anti-malaria or anti-virus effectors. The molecular mechanisms involved in the interactions between Asaia and mosquito hosts, as well as Asaia and other bacterial members of the mosquito microbiome, remained unexplored. Here, we determined the genome sequence of the strain W12 isolated from Anopheles stephensi mosquitoes, compared them to other Asaia species associated with plants or insects, and investigated some properties of the bacteria relevant to their symbiosis with host mosquitoes. The assembled genome of strain W12 has a size of 3.94 MB, which is the largest among Asaia spp studied so far. At least 3,585 coding sequences were predicted. The insect-associated Asaia including strain W12 carried more glycoside hydrolase (GH) encoding genes (31 per genome) than those isolated from plants (22 per genome). W12 had the most predicted regulatory protein components (213) among the selected Asaia (ranging from 131 to 211), indicating its great capability to adapt to frequent environmental changes in the mosquito gut. Two complete operons encoding cytochrome bo3-type ubiquinol terminal oxidases (cyoABCD-1 and cyoABCD-2) were found in most of Asaia genomes, which possibly offer alternative terminal oxidases and allow the flexible transition of respiratory pathways. Genes involved in the production of acetoin and 2,3-butandiol have been identified in Asaia sp. W12.

In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.