FIG. 3.

Substrates protect AcnB against iron loss and oxidation. (A) Concentrated extracts containing AcnB (JRG2789) were diluted to 0.25 mg/ml anaerobically in 50 mM Tris-HCl without or with 30 mM citrate or 30 mM isocitrate. For the latter two samples, the loss of activity was deduced from the rate of the ongoing reaction. The activity of the sample lacking substrate was determined at indicated time points by removal and assay of aliquots. Over the same period of time, concentrated enzyme lost no activity (data not shown). (B) Concentrated anaerobic extracts of SMV3 (AcnB⁺ AcnA⁻ SOD⁻) were exposed to air with or without 30 mM citrate or 30 mM isocitrate. Aconitase activity was determined at the indicated time points. Representative experiments of >3 replicates are shown; assay variability was <10%.