Anaerobically grown Escherichia coli cells were shown to acidify the reaction medium in response to oxygen or dimethyl sulfoxide (DMSO) pulses, with the H ؉ /e ؊ stoichiometry being close to 2.5 and 1.5, respectively. In the presence of the NADH dehydrogenase I (NDH-I) inhibitor 8-methyl-N-vanillyl-6-nonenamide (capsaicin) or in mutants lacking NDH-I, this ratio decreased to 1 for O 2 and to 0 for DMSO. These data suggest that (i) the H ؉ /e ؊ stoichiometry for E. coli NDH-I is at least 1.5 and (ii) the DMSO reductase does not generate a proton motive force.NADH oxidation by the respiratory chain of Escherichia coli is catalyzed via at least two distinct NADH:quinone oxidoreductases, i.e., NADH dehydrogenase I (NDH-I) and NADH dehydrogenase II (NDH-II) (5, 15). NDH-II comprises a single polypeptide containing flavin adenine dinucleotide, and this enzyme cannot form ⌬ H ϩ (31). NDH-I consists of 14 individual subunits containing flavin mononucleotide and five to eight FeS clusters (12,21,24); its NADH-oxidizing activity is coupled to ⌬ H ϩ formation. The operon encoding NDH-I was cloned and sequenced (24), and the protein was purified and characterized (12). The sequence and the set of prosthetic groups of this enzyme closely resemble the mitochondrial complex I. In fact, E. coli NDH-I is considered to be a simplified version of this type of enzyme or a so-called minimal form of complex I.The H ϩ /e Ϫ stoichiometry of the mitochondrial complex I is usually assumed to be 2 (see, e.g., references 17 and 27), although values of 1.5 (23) and 2.5 (13) have also been discussed. The H ϩ /e Ϫ stoichiometry for NDH-I from E. coli still awaits elucidation, mainly because of the wide diversity of NDH-I-independent respiratory chain-linked dehydrogenases present in this bacterium. These enzymes reduce quinone without ⌬ H ϩ generation, which can result in a decrease in the measured H ϩ /e Ϫ ratio for NDH-I. To overcome this difficulty, one can use E. coli cells growing anaerobically. In such cells, synthesis of all the noncoupled dehydrogenases is strongly repressed by the Arc and Fnr regulatory systems (14), while the expression of NDH-I is decreased only slightly (4). Anaerobic growth of E. coli can also be accompanied by the appearance of a number of additional membrane-bound dehydrogenases (anaerobic glycerophosphate dehydrogenase, formate dehydrogenase, and hydrogenase) and a wide variety of reductases such as fumarate reductase, trimethylamine N-oxide (TMAO) reductase, dimethyl sulfoxide (DMSO) reductase, nitrate reductase, and nitrite reductase (8).The DMSO reduction in anaerobic cultures of E. coli is catalyzed by an enzyme (DMSO reductase) which is composed of three different subunits containing four FeS clusters and a molybdopterin cofactor (1,19). DMSO reduction by anaerobically grown E. coli is accompanied by H ϩ extrusion from the cells with H ϩ /e Ϫ stoichiometry close to 1.5 (3). It was unknown whether any respiratory chain enzymes that might precede DMSO reductase (first of all, NDH-I) are involved in ⌬ H ϩ fo...
The aeration-dependent changes in content of various quinones in Escherichia coli were found to be unaffected by a prokaryotic translation inhibitor chloramphenicol. In addition, this process was shown to be completely intact in cells with mutated fnr, arc and appY loci. It is assumed that E. coli possesses a special system of oxygen-dependent post-transcriptional regulation of the quinone biosynthetic pathways.
Growth of .E. coli in the presence of the protonophorous uncoupler pentachlorophenol is shown to strongly enhance levels of cytochrome d, a putative Na+-motive oxidase. This effect was found to be arrested by chloramphenicol and stimulated by high Na+ concentration in the growth medium. The induction of cytochrome d takes place in a mutant deficient in the FoF, ATP-synthase but does not occur in mutants deficient in either of two different components of the Arc system. Similar relationships were revealed when pentachlorophenol was replaced by ferricyanide and phenazine methosulfate, agents oxidizing the respiratory chain. Induction of cytochrome dis also shown to occur in riboflavin-deficient mutants growing in the presence of such low riboflavin concentrations as to be insutlicient to maintain a high respiration rate. It is suggested (i) that it is &+ decrease rather than reduction of the respiratory chain that is the signal for the induction of cytochrome d, and (ii) the Arc system is involved in this type of metabolic regulation.
Regulation of synthesis of cytochrome d in Escherichia coli has been studied using mutants with cytochrome‐d‐β‐galactosidase gene fusions. It was shown that various protonophorous uncouplers, when added to the growth medium, cause induction of the cytochrome d synthesis. The cytochrome‐d‐inducing activity of uncouplers correlates with their ability to inhibit such a ΔH+‐driven function as motility of the E. coli cells. An increase in the Na+ concentration in the growth medium from 1.5mM to 25mM results in induction of the cytochrome d synthesis. The cytochrome‐d‐inducing effect of uncouplers is much more pronounced when the Na+ concentration is high than when it is low. These data are in agreement with the assumption that cytochrome d is involved in the Na+ energetics substituting for the H+ energetics when the latter appears to be inefficient. Mutations in arcA or arcB genes (but not in fnr gene) completely prevent the increase in the cytochrome d level induced by uncouplers but are without effect on that induced by Na+. It is assumed that in the control of the cytochrome d synthesis, the Arc system is involved in the ΔH+ sensing whereas sensing of Δ;Na+ (or of the Na+ concentration) is mediated by some other receptor system.
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