Respiration of Escherichia coli Can Be Fully Uncoupled via the Nonelectrogenic Terminal Cytochrome bd-II Oxidase

The respiratory chain of Escherichia coli is usually considered a device to conserve energy via the generation of a proton motive force, which subsequently may drive ATP synthesis by the ATP synthetase. It is known that in this system a fixed amount of ATP per oxygen molecule reduced (P/O ratio) is not synthesized due to alternative NADH dehydrogenases and terminal oxidases with different proton pumping stoichiometries. Here we show that P/O ratios can vary much more than previously thought. First, we show that in wild-type E. coli cytochrome bo, cytochrome bd-I, and cytochrome bd-II are the major terminal oxidases; deletion of all of the genes encoding these enzymes results in a fermentative phenotype in the presence of oxygen. Second, we provide evidence that the electron flux through cytochrome bd-II oxidase is significant but does not contribute to the generation of a proton motive force. The kinetics support the view that this system is as an energy-independent system gives the cell metabolic flexibility by uncoupling catabolism from ATP synthesis under non-steady-state conditions. The nonelectrogenic nature of cytochrome bd-II oxidase implies that the respiratory chain can function in a fully uncoupled mode such that ATP synthesis occurs solely by substrate level phosphorylation. As a consequence, the yield with a carbon and energy source can vary five- to sevenfold depending on the electron flux distribution in the respiratory chain. A full understanding and control of this distribution open new avenues for optimization of biotechnological processes.The aerobic respiratory chain of Escherichia coli can function with a variety of different membrane-bound NADH dehydrogenases, including NDH-I, NDH-II, and WrbA (8, 26-28), as well as YhdH and QOR (15, 38, 39), on the electron input side and three ubiquinol oxidases (cytochromes bd-I, bd-II, and bo) (12, 14, 19, 22, 29) on the output side (Fig. ​(Fig.1).1). The stoichiometry for the number of protons pumped for each two electrons transferred (H⁺/2e⁻ ratio) has unequivocally been determined for NDH-I (H⁺/2e⁻, 4) and NDH-II (H⁺/2e⁻, 0) (10, 23, 41). Although no specific data are available for WrbA, YhdH, and QOR, it is generally assumed that these NADH:quinone oxidoreductases are not electrogenic because of the absence of (predicted) transmembrane alpha-helices (15, 38, 39). Similarly, the energy-conserving efficiencies of the cytochrome bd-I oxidase and the cytochrome bo oxidase are different; the cytochrome bd-I complex does not actively pump protons, but due to the oxidation of the quinol on the periplasmic side of the membrane and subsequent uptake of protons from the cytoplasmic side of the membrane, which are used in the formation of water, net electron transfer results in proton translocation with an H⁺/2e⁻ stoichiometry of 2 (32). In contrast, the cytochrome bo complex actively pumps protons over the membrane, resulting in an H⁺/2e⁻ stoichiometry of 4 (33, 42). The stoichiometry of proton translocation of the cytochrome bd-II complex is unknown.Open in a separate windowFIG. 1.Diagram of all NADH:quinone oxidoreductases and quinol:oxygen oxidoreductases in E. coli and their proton translocation properties. Cyt, cytochrome; Q, quinone.Due to the differences in the H⁺/e⁻ ratios of the dehydrogenases involved, two-electron transfer from NADH to the quinone pool may be accompanied by the translocation of any number of protons between 0 and 4, and subsequent reoxidation of the quinol pool may contribute to proton translocation again with a stoichiometry that depends on the relative activities of the terminal oxidases. The loose coupling between energy conservation and electron flow in respiration has been interpreted as a physiological means for the cell to cope with sudden changes in the rate of electron influx into the respiratory chain and/or in the availability of terminal electron acceptors on its terminal side (10). The fact that this energetic efficiency can vary is of great interest, both for understanding the physiological adaptive responses of the microbial cell and for biotechnological applications (e.g., synthesis of any oxidized compound with minimal biomass production). For this, it is important to quantify the flux distribution over and the efficiencies of the components of the respiratory machinery in relation to environmental conditions.Previous studies (10) have shown that NDH-I, NDH-II, and the two well-characterized cytochrome oxidases contribute significantly to the overall electron flux and furthermore that the distribution of fluxes over these components depends on environmental conditions, such as the growth rate in glucose-limited chemostats (10). In addition, it has been suggested that the flux distribution over the terminal oxidases of E. coli is dependent on the culture pH (40). However, the cytochrome bd-II oxidase was not taken into account in these previous studies.Here we present data that show that cytochrome bd-II oxidase participates significantly in oxygen reduction both during nonlimited growth in batch cultures and in glucose-limited chemostat cultures. For further quantification of the contribution of the respiratory chain to oxidative phosphorylation, it is essential to assess the in vivo H⁺/2e⁻ stoichiometry of the cytochrome bd-II oxidase (4, 37). Essentially, the approach used in previous studies by Calhoun et al. (10) was followed: strains with respiratory chains that were modified such that their H⁺/2e⁻ stoichiometry was fixed and known were grown under identical, glucose-limited conditions in chemostat culture. A flux analysis with respect to glucose catabolism and respiration allowed calculation of the rate of ATP synthesis for these strains. The data were then used as reference flux data for a strain that contained the cytochrome bd-II oxidase as the sole terminal oxidase. This strain showed a decreased yield with respect to oxygen and glucose. In this way we demonstrated that electron flow through the cytochrome bd-II oxidase does not contribute to the generation of a proton motive force. The results are discussed in view of the biochemical characterization of the enzyme and its physiological importance to adaptive responses by E. coli to an ever-changing environment.