Fig. 3. Native MS of [4Fe–4S] FNR and the effect of O₂ exposure. (a) Deconvoluted mass spectrum of [4Fe–4S] S24F FNR before (black line) and after exposure (18 min, red line) to dissolved O₂. This resulted in the formation of a variety of protein-bound clusters including [3Fe–4S], [3Fe–3S] and [2Fe–2S] forms. Persulfide adducts of the [2Fe–2S] cluster and apo-FNR were also observed. (b) and (c) Temporal behaviours of the [4Fe–4S] cluster (black squares), [3Fe–4S] cluster (yellow triangles), [2Fe–2S] cluster (red diamonds) and apo FNR (white circles) forms (in b), and [2Fe–4S] cluster form (red diamonds) in (c) after O₂ exposure. Global fitting to the experimental data is shown as solid lines. See ref. 44 for further details. (d) Proposed mechanistic scheme for the conversion of [4Fe–4S] FNR and RirA based on ESI-MS data.^44,45 The initial [4Fe–4S] cluster is coordinated by three Cys residues. The fourth ligand, illustrated in the figure as “X”, is a Cys in FNR, but unknown for RirA. Both regulators follow a common [4Fe–4S] to [2Fe–2S] conversion pathway, via [3Fe–4S] and [3Fe–3S] clusters. The two mechanisms diverge at indicated branch points to give persulfide ligated [2Fe–2S] clusters (FNR, red box) or other FeS cluster types (RirA, black box).