Figure 6.

In vitro effects of CO on complex IV activity and reversal by Ngb-H64Q-CCC treatment. To demonstrate the specific effect of CO on complex IV activity in isolated rate mitochondria, a closed chamber with a Clark-like oxygen electrode was utilized. A combination of FCCP, TMPD, and ascorbate were added to demonstrate cytochrome c oxidase activity. A–D, four conditions were measured: baseline level of TMPD-ascorbate–driven respiration (labeled raw data, A), respiration after the addition of 200 μm CO in CO-saturated PBS followed by 100 μl of aerobic PBS (B), respiration after the addition of 100 μm chamber concentration of oxygenated-Ngb-H64Q-CCC (C), and finally, respiration after the addition of 200 μm CO in CO-saturated PBS followed by 100 μm of oxygenated-Ngb-H64Q-CCC (D). These rates were compared with the average rate of baseline TMPD-ascorbate–driven respiration for a given day of experiments and the same animal. E, the ratios between experimental arms were compared. In these experiments, the addition of Ngb-H64Q-CCC itself slowed complex IV activity to 62 ± 7% of baseline. The addition of CO slowed complex IV activity to 24 ± 5% of baseline. The addition of Ngb-H64Q-CCC after CO lead to a respiration rate of 39 ± 6% of baseline, markedly higher than CO-exposed, untreated complex IV activity (unpaired Student's t test; ****, p < 0.0001). In an unmatched regular two-way ANOVA, we determined that there was a significant interaction between Ngb-H64Q-CCC and exposure to CO (interaction term; ****, p < 0.0001; single line) (F, representative raw traces compared). rNgb, Ngb-H64Q-CCC. #, Figs. 5A and 6A show the same control conditions, represented for clarity.