Downfield region of 1H NMR spectra of FeII–HNO WT GlbN. (A) A 1.5 mM FeIII
bis–histidine WT 15N GlbN sample (200 mM phosphate, pH 7.1, 10% D2O, 298 K, GODCAT) was treated with 7.5 mM MAHMA-NONOate. The resulting mixture of FeIII
bis–histidine and FeIII–NO GlbN was then treated with 7.5 mM DT. After ~ 50 min incubation, two FeII–HNO signals are detected (with major:minor intensity ratio ~ 4:1). The total HNO yield was estimated by peak integration to be ~ 23 %. (B) A 400 μM FeIII
bis–histidine WT GlbN sample (70 mM phosphate, pH 8.5 (pre DT), 10% D2O, 298 K, GODCAT) incubated with 5 mM 15N-nitrite was treated with 5 mM DT. After a ~ 2 h incubation, two H15NO doublets, each split by 1JNH = 71 Hz, were observed and correspond to FeII–H15NO GlbN (major signals) and FeII–H15NO GlbN-A (minor signals). The total H15NO yield was estimated by peak integration to be ~ 21 %.