Fig. 5. Effect of 1 mM EDTA on N8D RirA [4Fe–4S] cluster. (A) UV-visible absorbance spectra of N8D RirA recorded over several hours following the addition of 1 mM EDTA, under anaerobic conditions. The initial spectrum is in red, all intervening spectra are in grey, end point spectrum is in black (B) Plot of absorbance change obtained from anaerobic (black circles) and aerobic (red circles) experiments: A_{410 nm} was plotted as a function of time. (C) As in (A) but under aerobic conditions. The initial spectrum prior to O₂ exposure is a blue dashed line, the initial spectrum following exposure to O₂ is shown in red, while all intervening spectra are in grey, and the end point spectrum is in black. For both experiments, N8D RirA (29 μM in cluster, as isolated) was in 25 mM Hepes, 2.5 mM CaCl₂ 50 mM NaCl, 750 mM KCl, pH 7.5. Measurements were obtained using a 1 cm pathlength sealed anaerobic quartz cuvette.