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. 2017 Aug 1;83(16):e00867-17. doi: 10.1128/AEM.00867-17

FIG 4.

FIG 4

Copper inhibits iron-sulfur cluster assembly in E. coli cells under anaerobic conditions. (A) UV-visible absorption spectra of recombinant fumarase B purified from E. coli cells. Spectrum 1, fumarase B was expressed in the exponentially growing E. coli cells in LB medium without copper addition under anaerobic conditions for 2 h; spectrum 2, fumarase B was expressed in the exponentially growing E. coli cells in LB medium supplemented with 0.2 mM CuCl2 under anaerobic conditions for 2 h; spectrum 3, fumarase B was expressed in the exponentially growing E. coli cells in LB medium under anaerobic conditions for 2 h, followed by addition of 0.2 mM CuCl2 and an additional 2 h of growth under anaerobic conditions. Without addition of copper, fumarase B expressed in E. coli cells for 4 h had an absorption spectrum very similar to that of spectrum 3. The protein concentration of fumarase B was 20 μM. (Inset) Photograph of SDS-PAGE gel of purified proteins. (B) Relative activities of purified fumarase B from panel A. For enzyme activity assay, 10 nM fumarase B was used. The highest enzyme activity (100%) for fumarase B was 22.7 ± 0.8 μΜ/μM/min. (C) UV-visible absorption spectra of recombinant endonuclease III (Nth) purified from E. coli cells. Spectrum 1, endonuclease III was expressed in the exponentially growing E. coli cells in LB medium without copper addition under anaerobic conditions for 2 h; spectrum 2, endonuclease III was induced in the exponentially growing E. coli cells in LB medium supplemented with 0.2 mM CuCl2 under anaerobic conditions for 2 h; spectrum 3, endonuclease III was expressed in the exponentially growing E. coli cells in LB medium under anaerobic conditions for 2 h, followed by addition of 0.2 mM CuCl2 and an additional 2 h of growth under anaerobic conditions. Without addition of copper, endonuclease III expressed in E. coli cells for 4 h had an absorption spectrum very similar to that of spectrum 3. The protein concentration of endonuclease III was 40 μM. (Inset) Photograph of the SDS-PAGE gel of purified proteins.