Fig. 5: Effects of NO-ferroheme on mitochondria and apo-sGC activation.
a,b, The effect of NO-ferroheme-Mb and the NO donor DEA-NONOate on mitochondrial (CI + CII) state 3 respiration evaluated by high-resolution respirometry. a, DEA-NONOate clearly inhibited mitochondrial respiration, whereas NO-ferroheme-Mb had no inhibitory effect. b, Original tracings showing mitochondrial respiration in an oxygraph and the effects of DEA-NONOate (1 μM, top) and Mb-NO (1 μM, middle and 10 μM, bottom). DEA-NONOate completely inhibited (CI + CII-dependent) state 3 respiration (red curve, top), whereas NO-ferroheme-Mb had no effect (red curve, middle and bottom). Blue curves represent oxygen concentration in the chamber. c, Apo-sGC was prepared and supplemented with either NO-ferroheme-Mb (10 μM) or DEA-NONOate (10 μM), followed by an analysis of cGMP production using an ELISA kit. Apo-sGC was clearly activated by NO-ferroheme-Mb, but not by NO released from DEA-NONOate. Respirometry data in a were repeated three times and analyzed by paired two-way repeated measures ANOVA followed by Å Ãdák’s multiple comparisons test. Data in c were analyzed by nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Dots connected with lines in a represent paired data from three independent experiments. Dots in c represent the number of independent observations, and the data are presented as mean ± s.d. a,c, P values for statistically significant differences are indicated. ****P ≤ 0.0001. Differences not significant are indicated as NS.
