Fig. 2.

The Mtb WhiB4 Fe-S cluster responds to O₂ and NO. A. UV-visible spectra were obtained before and after exposing anaerobically reconstituted WhiB4 4Fe-4S cluster to air at various time points. Note the time-dependent decrease in the absorbance at 420 nm. B. Air-exposed samples of reconstituted WhiB4 were withdrawn immediately (5 min; green spectrum) or 30 min post exposure (blue spectrum) and analysed by EDFS-EPR. The appearance of a sharp signal at g = 2.01 indicates a [3Fe-4S]¹⁺ cluster. Conditions for EPR spectroscopy were the same as in Fig. 1C. The influence of NO on WhiB4 was analysed by adding a 10-fold molar excess of proline NONOate (WhiB4:NO) before analysis by UV-visible and cw-EPR spectroscopy. C. UV-visible spectra were acquired before and after addition of proline NONOate. Note the increase in the characteristic monomeric DNIC peak at ∼ 350 nm in the NO-treated sample. D. cw-EPR spectra of NO-treated samples were acquired at a microwave frequency of 9.667 GHz and microwave power of 2 mW at 100, 155 and 200 K. The appearance of a sharp signal around 2.03 and the increase in the intensity of the signal at lower temperatures is consistent with the formation of monomeric DNIC.