Uncoupling of substrate-level phosphorylation in Escherichia coli during glucose-limited growth

Abstract

The respiratory chain of Escherichia coli contains three different cytochrome oxidases. Whereas the cytochrome bo oxidase and the cytochrome bd-I oxidase are well characterized and have been shown to contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochrome bd-II oxidase. Recently, however, this view was challenged by an in vitro biochemical analysis that showed that the activity of cytochrome bd-II oxidase does contribute to proton translocation with an H(+)/e(-) stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growing E. coli strains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochrome bd-II oxidase, compatible with an H(+)/e(-) ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation in E. coli under energy-limited growth conditions.

Fig 1

Scheme of the E. coli aerobic respiratory chain (adapted from reference with permission from the publisher). Enzyme bioenergetic efficiency is indicated as the number of protons delivered to the periplasmic side of the membrane per electron (the H⁺/e⁻ ratio). NDH I is the proton-translocating NADH-quinone oxidoreductase, and NDH II, WrbA, QOR, QOR2, and YhDH are the non-proton-translocating NADH-quinone oxidoreductases (NP NDHs). NDH I and the NP NDHs transfer electrons to the quinones (Q) to yield reduced quinones (QH₂). Three quinol-oxygen oxidoreductases, cytochromes bo₃ (Cybo), bd-I (Cybd I), and bd-II (Cybd II), oxidize QH₂ and reduce O₂ to H₂O. Acetate formation via acetate kinase (AcK) leads to ATP formation, in contrast to acetate synthesized by PoxB. Similarly, lactate formed by one of the three lactate dehydrogenases results in ATP formation (LDH here represents Dld, LdhA, and LldD), while lactate synthesized via methylglyoxal synthase (MGOS) from DHAP leads to ATP consumption via the activity of the phosphotransferase system (PTS) and 6-phosphofructokinase (PFK). The gray area indicates the cytoplasmic membrane. The dashed and dotted lines indicate uncoupling at the level of substrate phosphorylation catabolic pathways by MGOS and PoxB, respectively.