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. 2019 Sep 17;8:e47804. doi: 10.7554/eLife.47804

Figure 8. EPR analysis of [4Fe-4S] RirA cluster conversion under aerobic and anaerobic low iron conditions.

(A) EPR spectra of [4Fe-4S] RirA at specified time points following the addition of 250 µM EDTA under aerobic conditions at 37°C. (B) Plot of g = 2.01 EPR signal intensity dependence on temperature at a range of microwave powers. The dashed red line represents a hyperbolic 1/T function plotted in arbitrary units to represent the Curie law (when no saturation effects take place). All experimental dependences are individually scaled so that each meets the hyperbola via at least one data point, with remaining data points lying below the hyperbola (indicating that the signal saturates). (C) Plot of ESI-MS relative abundances of RirA [3Fe-4S] cluster as a function of time following exposure of RirA [4Fe-4S] to 250 µM EDTA at 37° C and aerobic conditions (yellow triangles) and RirA [3Fe-4S]1+ cluster determined under identical condition by EPR under aerobic (+O2, white-filled circles) and anaerobic (-O2, blue-filled circles) conditions. The solid yellow line represents the global fit to the ESI-MS data (as shown in Figure 7). (D) As in (A) but under anaerobic conditions. [4Fe-4S] RirA (~25 µM) was in aerobic (~228 µM O2) 250 mM ammonium acetate pH 7.3.

Figure 8.

Figure 8—figure supplement 1. EPR saturation characteristics of the RirA EPR signal.

Figure 8—figure supplement 1.

A complete temperature/microwave power saturation map of the RirA EPR signal (at 3 min after aerobic addition of EDTA to RirA). All spectra within each panel are plotted at the same receiver gain, and the relative gain between panels is indicated by the ‘x’ symbol. The broad line at g = 2.05 (to the left from the main signal) results from a minor baseline copper EPR signal. Instrumental settings were as follows: microwave frequency νMW = 9.4647 GHz; modulation frequency νm = 100 kHz; modulation amplitude Am = 3 G; time constant τ = 41 ms; scan rate v = 8.34 G/s.
Figure 8—figure supplement 2. Temperature dependences of the RirA EPR signal at different microwave powers.

Figure 8—figure supplement 2.

Plots of relative intensity (as determined from signals’ first integral maximal value which were normalised to signal intensity of 1.00) at six selected values of microwave power, as indicated.