Figure 6.

Glutathione is an important intracellular and extracellular target of CORM-2 (I–IV) and CORM-2 has a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) as investigated by ¹H NMR (V–VIII). In I, the effects are shown of extracellular reduced glutathione (GSH) or oxidised glutathione (GSSG). The grey line shows cell viability with CORM-2 alone. GSH fully restored viability to that of the no-CORM control (dashed line). II shows CORM-induced intracellular Ru accumulation in the absence or presence of GSH or GSSG (* p ≤ 0.05, ** p ≤ 0.01, assessed via unpaired t-test). CORM-2 stocks were incubated with a 2-fold excess of the compound prior to addition of 30 µM CORM-2. (III) Uptake of CORM-2-derived Ru is significantly reduced in a glutathione-deficient mutant (gshA, open symbols, dashed line) compared to the GshA⁺ wild-type parent strain (gshA⁺, solid line) (* p ≤ 0.05, *** p ≤ 0.001, assessed via unpaired t-test). (IV) Culture viability (CFU mL⁻¹) of the glutathione-deficient strain compared to the parent strain following addition of 7.5 or 15 µM CORM-2. Data in I–IV represent 3 biological repeats ± SD. (V) NMR spectra of the Cys Hβ resonances of GSH (1 mM) on titration with 0–5 mM CORM-2 and (VI) corresponding binding curve. (VII) NMR spectra of the Cys Hβ resonances of GSSG (0.5 mM) on titration with 0–14.5 mM CORM-2 and (VIII) corresponding binding curve. In each titration the arrow shows the direction of increasing [CORM-2]. The estimated K_d of CORM-2 to GSH was determined to be: 2 ± 1 μM for GSH (VI) and 25 ± 5 mM for GSSG (VIII). Titrations were performed in 30 mM KPi buffer pH 7.4 and pH was maintained by adjustment with KOH throughout the experiment.