Table 1.

Aconitase and IRE binding functions of purified S138 IRP1 mutants

IRP1	Reconstituted aconitase activity, units/mg	IRE binding affinity of apo-IRP1, pM
wt	19.8  ±  3.5	61  ±  19
S138A	24.3  ±  1.5	23  ±  6.3
S138D	18.5  ±  1.1	36  ±  18.5
S138E	10.8  ±  1.8	19  ±  7

The various IRP1 mutants were purified from yeast and subjected to anaerobic Fe–S cluster reconstitution followed by aconitase assay, or the apoprotein was analyzed for IRE-binding ability. Reconstitution and analysis of aconitase activity was performed on a minimum of three independent preparations of each mutant IRP1. Saturation curves to determine the affinity of IRP1–IRE interaction were done a minimum of four times representing at least two independent experiments for each mutant IRP1. Values shown are ± SD.