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The Terminal Oxidase Cytochrome bd Promotes Sulfide-resistant Bacterial Respiration and Growth.

Forte E, Borisov VB, Falabella M, Colaço HG, Tinajero-Trejo M, Poole RK, Vicente JB, Sarti P, Giuffrè A - Sci Rep (2016)

Bottom Line: E. coli has three respiratory oxidases, the cyanide-sensitive heme-copper bo3 enzyme and two bd oxidases much less sensitive to cyanide.In E. coli respiratory mutants, both O2-consumption and aerobic growth proved to be severely impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected by ≤200 μM sulfide when either bd enzyme acted as the only terminal oxidase.The impact of this discovery is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Sciences and Istituto Pasteur- Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy.

ABSTRACT
Hydrogen sulfide (H2S) impairs mitochondrial respiration by potently inhibiting the heme-copper cytochrome c oxidase. Since many prokaryotes, including Escherichia (E.) coli, generate H2S and encounter high H2S levels particularly in the human gut, herein we tested whether bacteria can sustain sulfide-resistant O2-dependent respiration. E. coli has three respiratory oxidases, the cyanide-sensitive heme-copper bo3 enzyme and two bd oxidases much less sensitive to cyanide. Working on the isolated enzymes, we found that, whereas the bo3 oxidase is inhibited by sulfide with half-maximal inhibitory concentration IC50 = 1.1 ± 0.1 μM, under identical experimental conditions both bd oxidases are insensitive to sulfide up to 58 μM. In E. coli respiratory mutants, both O2-consumption and aerobic growth proved to be severely impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected by ≤200 μM sulfide when either bd enzyme acted as the only terminal oxidase. Accordingly, wild-type E. coli showed sulfide-insensitive respiration and growth under conditions favouring the expression of bd oxidases. In all tested conditions, cyanide mimicked the functional effect of sulfide on bacterial respiration. We conclude that bd oxidases promote sulfide-resistant O2-consumption and growth in E. coli and possibly other bacteria. The impact of this discovery is discussed.

No MeSH data available.


Related in: MedlinePlus

NaHS inhibition of isolated cytochrome bo3.Percentage inhibition of the O2 reductase activity of isolated cytochrome bo3 (6 nM) measured at increasing concentration of NaHS, in the presence of the 10 mM DTT and 0.25 mM Q1.
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f2: NaHS inhibition of isolated cytochrome bo3.Percentage inhibition of the O2 reductase activity of isolated cytochrome bo3 (6 nM) measured at increasing concentration of NaHS, in the presence of the 10 mM DTT and 0.25 mM Q1.

Mentions: The effect of sulfide on the O2 reductase activity of the E. coli respiratory oxidases, cytochromes bo3, bd-I and bd-II, was initially investigated testing the ability of each purified oxidase to consume O2 before and after addition of the sulfide donor NaHS. In these assays, O2 consumption was measured in the presence of dithiothreitol (DTT) and 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone (Q1) as the reducing system. As shown in Fig. 1A, NaHS (~7 μM) rapidly and effectively inhibits the O2 reductase activity of the isolated cytochrome bo3. The enzyme is inhibited with an apparent half-maximal inhibitory concentration IC50 = 1.1 ± 0.1 μM (Fig. 2). The inhibition of cytochrome bo3 is fully reversible. A rapid and complete recovery of the O2 reductase activity of the isolated enzyme was observed, when sulfide was quickly removed from solution by addition of an excess of O-acetyl-L-serine (OAS) and catalytic amounts of the sulfide-consuming O-acetylserine sulfhydrylase enzyme from Entamoeba histolytica (EhOASS, Fig. 1A). Sulfide consumption by EhOASS in the presence of OAS was assessed independently using a H2S-selective electrode (Figure S1). Notably, while being an effective inhibitor of E. coli cytochrome bo3, NaHS proved to be unable to inhibit the two E. coli bd-type oxidases. Addition of NaHS, even at high concentration (58 μM), did not alter the O2 consumption catalyzed by the bd-I or bd-II enzyme in the presence of DTT and Q1 (Fig. 1A). No O2 consumption stimulation by the OAS/EhOASS sulfide-scavenging system was observed in control oxygraphic experiments carried out in the absence or presence of the isolated oxidases (not shown).


The Terminal Oxidase Cytochrome bd Promotes Sulfide-resistant Bacterial Respiration and Growth.

Forte E, Borisov VB, Falabella M, Colaço HG, Tinajero-Trejo M, Poole RK, Vicente JB, Sarti P, Giuffrè A - Sci Rep (2016)

NaHS inhibition of isolated cytochrome bo3.Percentage inhibition of the O2 reductase activity of isolated cytochrome bo3 (6 nM) measured at increasing concentration of NaHS, in the presence of the 10 mM DTT and 0.25 mM Q1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4815019&req=5

f2: NaHS inhibition of isolated cytochrome bo3.Percentage inhibition of the O2 reductase activity of isolated cytochrome bo3 (6 nM) measured at increasing concentration of NaHS, in the presence of the 10 mM DTT and 0.25 mM Q1.
Mentions: The effect of sulfide on the O2 reductase activity of the E. coli respiratory oxidases, cytochromes bo3, bd-I and bd-II, was initially investigated testing the ability of each purified oxidase to consume O2 before and after addition of the sulfide donor NaHS. In these assays, O2 consumption was measured in the presence of dithiothreitol (DTT) and 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone (Q1) as the reducing system. As shown in Fig. 1A, NaHS (~7 μM) rapidly and effectively inhibits the O2 reductase activity of the isolated cytochrome bo3. The enzyme is inhibited with an apparent half-maximal inhibitory concentration IC50 = 1.1 ± 0.1 μM (Fig. 2). The inhibition of cytochrome bo3 is fully reversible. A rapid and complete recovery of the O2 reductase activity of the isolated enzyme was observed, when sulfide was quickly removed from solution by addition of an excess of O-acetyl-L-serine (OAS) and catalytic amounts of the sulfide-consuming O-acetylserine sulfhydrylase enzyme from Entamoeba histolytica (EhOASS, Fig. 1A). Sulfide consumption by EhOASS in the presence of OAS was assessed independently using a H2S-selective electrode (Figure S1). Notably, while being an effective inhibitor of E. coli cytochrome bo3, NaHS proved to be unable to inhibit the two E. coli bd-type oxidases. Addition of NaHS, even at high concentration (58 μM), did not alter the O2 consumption catalyzed by the bd-I or bd-II enzyme in the presence of DTT and Q1 (Fig. 1A). No O2 consumption stimulation by the OAS/EhOASS sulfide-scavenging system was observed in control oxygraphic experiments carried out in the absence or presence of the isolated oxidases (not shown).

Bottom Line: E. coli has three respiratory oxidases, the cyanide-sensitive heme-copper bo3 enzyme and two bd oxidases much less sensitive to cyanide.In E. coli respiratory mutants, both O2-consumption and aerobic growth proved to be severely impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected by ≤200 μM sulfide when either bd enzyme acted as the only terminal oxidase.The impact of this discovery is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Sciences and Istituto Pasteur- Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy.

ABSTRACT
Hydrogen sulfide (H2S) impairs mitochondrial respiration by potently inhibiting the heme-copper cytochrome c oxidase. Since many prokaryotes, including Escherichia (E.) coli, generate H2S and encounter high H2S levels particularly in the human gut, herein we tested whether bacteria can sustain sulfide-resistant O2-dependent respiration. E. coli has three respiratory oxidases, the cyanide-sensitive heme-copper bo3 enzyme and two bd oxidases much less sensitive to cyanide. Working on the isolated enzymes, we found that, whereas the bo3 oxidase is inhibited by sulfide with half-maximal inhibitory concentration IC50 = 1.1 ± 0.1 μM, under identical experimental conditions both bd oxidases are insensitive to sulfide up to 58 μM. In E. coli respiratory mutants, both O2-consumption and aerobic growth proved to be severely impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected by ≤200 μM sulfide when either bd enzyme acted as the only terminal oxidase. Accordingly, wild-type E. coli showed sulfide-insensitive respiration and growth under conditions favouring the expression of bd oxidases. In all tested conditions, cyanide mimicked the functional effect of sulfide on bacterial respiration. We conclude that bd oxidases promote sulfide-resistant O2-consumption and growth in E. coli and possibly other bacteria. The impact of this discovery is discussed.

No MeSH data available.


Related in: MedlinePlus