Figure 3. IRP1_S711E displays a profound defect in the first step of the aconitase catalytic reaction.

Purified recombinant wild-type IRP1 (white bars), IRP1_S711A (grey bars) or IRP1_S711E (black bars) (5 μg each) were analysed for aconitase activity after treatment with ferric citrate/sulphide to reconstitute the [4Fe-4S] cluster. (A) The conversion of citrate or cis-aconitate into isocitrate was measured by the coupled isocitrate dehydrogenase reaction [10]. (B) The formation of cis-aconitate from isocitrate was monitored spectrophotometrically at 240 nm [13]. (C) Schematic representation of the aconitase reaction, with the relative efficiency of IRP1_S711E to catalyse each step, compared with wild-type IRP1.