(A) Plots of relative abundances of [4Fe-4S] cluster (black) and [3Fe-4S] (yellow) species as a function of time following exposure to 250 µM EDTA at 37° C. Inset is a plot of relative abundances of the [4Fe-3S] cluster, illustrating that it is likely formed during ionisation. (B) – (E) As in (A) but showing [3Fe-3S] (B), [3Fe-2S] (C), [2Fe-2S] (D) and apo- (E) forms of RirA. Fits of the data, generated by a global analysis of the experimental data based on the reaction scheme depicted in Figure 5, are shown as solid lines. Broken lines correspond to the kinetic profile of the cluster species associated with that colour and are included to permit easy comparison between intermediates. Error bars show standard error for ESI-MS datasets (n = 4, derived from one biological replicate and three technical replicates). (F) Plot of A382 nm versus time following addition of 250 µM EDTA to [4Fe-4S] RirA (30 µM in cluster in 250 mM ammonium acetate, 500 µM glutathione, pH 7.3) at 37° C. The red line indicates a fit of the data generated using a bi-exponential function. We note that significant A382 nm remains after 30 min, where ESI-MS indicates that the majority of the protein is in an apo-form. The residual absorbance most likely arises from Fe/S species present in the cuvette, either attached to the protein, or in solution/suspension, for example as iron sulphide or iron acetate (Pellicer Martinez et al., 2017).