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. Author manuscript; available in PMC: 2016 Dec 3.
Published in final edited form as: J Biol Chem. 2006 Nov 13;282(2):929–937. doi: 10.1074/jbc.M607646200

Figure 3. H2O2 inactivates isopropylmalate isomerase by converting its [4Fe-4S]2+ cluster to a [3Fe-4S]+ cluster in vitro.

Figure 3

(A) An extract of anaerobically grown Hpx− cells was exposed to the indicated concentrations of H2O2 for 2 min at room temperature. Catalase then was added to remove H2O2. The experiment was conducted anaerobically. (B) Isopropylmalate isomerase from anaerobically cultured Hpx− cells was inactivated with 2 μM H2O2 for 5 min. Catalase was added to remove H2O2, and then 50 μM Fe(NH4)2(SO4)2 and 2.5 mM dithiothreitol were added. The activity was measured after 3 min incubation. (C) Hpx− cells that overproduce IPMI were harvested when the cells were OD600 of 0.2. The cell pellets were resuspended in 1/500 of original culture volume of 10% glycerol. The resuspended cells were incubated with 100 μM H2O2 for 1 min at 37°C. The cell suspension (250 μl) then was transferred into an EPR tube and frozen.