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. 2016 Mar 31:6:23788.
doi: 10.1038/srep23788.

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

Elena Forte  1 , Vitaliy B Borisov  2 , Micol Falabella  1 , Henrique G Colaço  3 , Mariana Tinajero-Trejo  4 , Robert K Poole  5 , João B Vicente  6 , Paolo Sarti  1 , Alessandro Giuffrè  7
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V体育2025版 - The Terminal Oxidase Cytochrome bd Promotes Sulfide-resistant Bacterial Respiration and Growth

Elena Forte et al. Sci Rep. .

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 VSports手机版. 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. .

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Figures

Figure 1
Figure 1. Effect of NaHS on E. coli terminal oxidases.
(A) O2 reductase activity of the isolated cytochromes bd-I (20 nM), bd-II (2.5 nM) and bo3 (6 nM) as measured at 25 °C in the presence of DTT (10 mM) and Q1 (0.25 mM). O2 consumption rates measured prior to NaHS addition (mean ± standard deviation, n = 3): 1.62 ± 0.07 μM O2/s (bd-I); 0.54 ± 0.02 μM O2/s (bd-II) and 0.46 ± 0.02 μM O2/s (bo3). O2 consumption by cytochrome bo3 is rapidly inhibited (~85%) by 7.2 μM NaHS, to be quickly and completely restored upon removal of sulfide from solution following the addition of 200 μM OAS and 216 nM EhOASS. On the contrary, NaHS (58 μM) does not affect the oxidase activity of either cytochrome bd-I or cytochrome bd-II. (B) O2 consumption by cell suspensions of the mutant strains expressing cytochrome bd-I (400 μl cells with OD600 = 1.85), cytochrome bd-II (600 μl cells with OD600 = 1.17) or cytochrome bo3 (200 μl cells with OD600 = 2.45), as the only terminal oxidase. O2 consumption rates measured prior to NaHS addition (mean ± standard deviation, n = 3): 0.20 ± 0.07 μM O2/s (bd-I); 0.18 ± 0.02 μM O2/s (bd-II) and 0.19 ± 0.02 μM O2/s (bo3). When sustained solely by cytochrome bo3, cell respiration is rapidly inhibited by 50 μM NaHS, to be quickly and completely restored following sulfide removal on addition of OAS (200 μM) and EhOASS (216 nM). No inhibition is observed following the addition of NaHS (50 μM), when respiration is sustained by the only cytochrome bd-I or bd-II.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. Effect of NaHS and cyanide on respiration of E. coli cells.
(Top) Residual respiratory activity measured after the addition of 50 μM NaHS to E. coli cells collected at the reported cell density. (Bottom) Comparison of the effect of cyanide and sulfide on cell respiration: respiratory activity measured after the addition of 50 μM NaHS or 50 μM NaCN to wild-type and mutant E. coli cells. Data (mean ± standard deviation) refer to the control activity measured before the addition of inhibitors (taken as 100%).
Figure 4
Figure 4. Effect of NaHS on E. coli cell growth.
Cell growth of E. coli wild-type (A) and mutant strains with bo3 (B), bd-I (C) or bd-II (D) as the only terminal oxidase, assayed in the presence (‘closed symbols’) or absence (‘open symbols’) of 200 μM NaHS. Inset to panel B: Effect of NaHS on the growth of the bo3-only expressing mutant, as evaluated at 2 hours after addition of NaHS used at the indicated concentrations. ‘Relative OD’ indicates the ratio between the optical density measured at 600 nm in the presence of NaHS and the one recorded after the same period of time (2 hours) in the absence of NaHS. Data expressed as mean ± standard deviation.
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