Electrochemical proton gradient in inverted membrane vesicles from Escherichia coli

Abstract

Inverted membrane vesicles prepared from Escherichia coli ML 308-225 generate a transmembrane electrochemical proton gradient (delta mu H+; interior positive and acid) during oxidation of D-lactate, succinate, reduced phenazine methosulfate, or NADH or hydrolysis of ATP. Using the distribution of the lipophilic anion thiocyanate to measure the membrane potential (delta psi) and the permeant weak base methylamine to measure the pH gradient (delta pH), maximal values for delta psi H+ of approximately +160 mV are obtained. Many of the properties of delta psi H+ in inverted vesicles are similar to those described previously in right-side-out vesicles [Ramos, S., & Kaback, H.R. (1977) Biochemistry 16, 848]: (1) the magnitude of the delta psi (interior positive) generated in the presence of D-lactate or reduced phenazine methosulfate is similar to that observed in right-side-out vesicles but of opposite polarity and independent of pH from 5.5 to 8.0; (2) plots of delta pH vs. internal pH in the right-side-out vesicles are similar with D-lactate as the electron donor; (3) as observed with right-side-out vesicles, dissipation of delta psi or delta pH leads to a concomitant increase in the other parameter without a change in the rate of respiration; (4) inverted vesicles catalyze Na+ accumulation, and it is apparent that the process can be driven by either delta psi (interior positive) or delta pH (interior acid).

A Self‐Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone‐10 Cycling between Complex I and the Alternative Oxidase

“…15 Second, the respiratory control ratio (RCR) is the ratio of the coupled and uncoupled rates of catalysis: Δ p opposes catalysis, so when an “uncoupler” is used to dissipate Δ p , the rate increases. The RCR value for NADH oxidation by Q 10 PLs (1.1, 4 H + translocated per NADH) is low compared to values reported for submitochondrial particles (3.0–5.5,11, 12, 16 10 H + per NADH for complexes I, III, and IV) and complex I containing PLs measured using DQ (2.2–4.5,17, 18 4 H + per NADH) However, lower values are typical for succinate oxidation by SMPs (1.6–3.2,11, 12, 16 6 H + per succinate from complexes II, III and IV), and the highest value from inverted Escherichia coli vesicles that are known to sustain Δ p ≈160 mV19 is 1.720 (6 H + per NADH, from complex I and an ubiquinol oxidase). Higher RCR values are usually taken to indicate “better coupled” vesicles, but the values are also affected by the enzyme activity and configuration.…”

The Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Is a Primary Proton Pump but May Be Capable of Secondary Sodium Antiport

“…This transport was inhibited by the addition of EIPA, an inhibitor of Na ϩ /H ϩ antiporters (21). However, data obtained with E. coli wild-type cells and membrane vesicles indicated the presence of an NADH-induced proton gradient (10,22,23). In addition, FT-IR spectroscopy has shown that the redox reaction of the E. coli complex I is associated with the protonation of tyrosines and acidic amino acids (24,25).…”

The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions

“…E. coli UTL2mdfA::kan cells overexpressing MdfA were grown and treated as described (15). Inverted membrane vesicles were prepared from these cells as described (18). Cells were washed once in 50 mM potassium phosphate, pH 7.5͞5 mM MgSO 4 .…”