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. 2012 Nov 16;586(22):4016-22.
doi: 10.1016/j.febslet.2012.10.001. Epub 2012 Oct 12.

The E. coli SufS-SufE sulfur transfer system is more resistant to oxidative stress than IscS-IscU

Yuyuan Dai  1 , F Wayne Outten
Affiliations

The E. coli SufS-SufE sulfur transfer system is more resistant to oxidative stress than IscS-IscU

Yuyuan Dai et al. FEBS Lett. .

Abstract

During oxidative stress in Escherichiacoli, the SufABCDSE stress response pathway mediates iron-sulfur (Fe-S) cluster biogenesis rather than the Isc pathway. To determine why the Suf pathway is favored under stress conditions, the stress response SufS-SufE sulfur transfer pathway and the basal housekeeping IscS-IscU pathway were directly compared. We found that SufS-SufE cysteine desulfurase activity is significantly higher than IscS-IscU at physiological cysteine concentrations and after exposure to H(2)O(2). Mass spectrometry analysis demonstrated that IscS-IscU is more susceptible than SufS-SufE to oxidative modification by H(2)O(2). These important results provide biochemical insight into the stress resistance of the Suf pathway.

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Figures

Figure 1
Figure 1
Kinetic analysis of SufS activity in response to varied substrate concentrations. The reactions contained (A) 0.5 µM SufS, 4 µM SufE, 2 mM DTT and 10 – 500 µM L-cysteine or (B) 0.5 µM SufS, 0 – 15 µM SufE, 2 mM DTT, and 2 mM L-cysteine. The lines are the best fits to the Michaelis – Menten equation obtained using GraphPad Prism.
Figure 2
Figure 2
Substrate inhibition of SufS by SufE at lower concentrations of L-cysteine. (A) The reactions contain 0.5 µM SufS, 0 – 10 µM SufE, 2 mM DTT, and 10 – 20,000 µM L-cysteine (see embedded legend). (B) The reactions contain 0.5 µM SufS, 50 µM Cysteine, 2 mM DTT, 4 µM (●) or 8 µM (◆) SufE with increasing concentrations of SufBC2D (0 – 4 µM). A control reaction with 2 mM Cysteine, 2 mM DTT, 0.5 µM SufS, and 8 µM SufE with increasing concentrations of SufBC2D (0 – 4 µM) is also shown (■). Double reciprocal plots of kinetic data. Activity of 0.5 µM SufS, 2 mM DTT, and (C) varied 10 – 20,000 µM L-cysteine at several fixed concentrations of SufE or (D) varied 0.1 – 10 µM SufE at several fixed concentrations of L-cysteine. See embedded legend for symbol explanations.
Figure 3
Figure 3
Direct activity comparison of the SufS-SufE and IscS-IscU sulfur transfer systems. SufSSufE activity was divided by IscS activity (closed circles ●) or the IscS-IscU activity (open circles ○) and the ratios were plotted as a function of the L-cysteine concentration in the reaction. The reactions contain 0.5 µM SufS or IscS, 1.5 µM SufE or IscU, and 0.03 – 10 mM L-cysteine and DTT.
Figure 4
Figure 4
The sensitivity of SufS-SufE and IscS-IscU to H2O2 during the cysteine desulfurase reaction. 1 µM SufS or IscS and (where indicated) 10 µM SufE or IscU were mixed for 5 min. 2 mM L-cysteine was added to initiate the reaction followed immediately by 0 – 400 µM H2O2. After 30 minutes the reaction was quenched by heating at 95 °C for 5 minutes, followed by the addition of 2 mM DTT to reduce and release sulfide for measurement as described in Supplementary Materials Methods. All steps were carried out anaerobically. (A) Desulfurase activity of SufS (□), IscS (●), IscS-IscU (■) and SufS-SufE (◆). (B) Percent activity of IscS (black bar), IscS-IscU (light grey bar), and SufS-SufE (white bar) compared to their activity without H2O2.
Figure 5
Figure 5
(A) Percent oxidation of active site Cys residues in the IscS, IscU, SufS, and SufE proteins after H2O2 exposure during the cysteine desulfurase reaction. (B) and (C) Reducing and non-reducing 12% SDS-PAGE gel separation of H2O2 treated proteins. The proteins were treated the same way as the samples for mass spectrometry analysis (see text). Proteins were precipitated with 10% TCA and dissolved in 1 X SDS loading buffer with or without DTT. Samples were heated at 95 °C for 10 min before loading on the gel. (B) IscU and IscS-IscU gel separation. (C) SufE and SufS-SufE gel separation.
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