Figure 4.

Amphidirectional CII operation during acute anoxia. (A) Scheme illustrating the action of inhibitor(s) used downstream of CI in the experiments shown in the figure (created with BioRender.com). Quantification of rotenone-induced changes in Q redox state (B–D) or NADH (E–G) signal. Eth (grey) ethanol, Rot rotenone, 1 μM (blue); S succinate, 5 mM (striped grey). (H) Untargeted metabolite analysis of succinate (S) present in the pellets of mitochondria treated with the conditions indicated in the panel. The y-axis illustrates normalized, log transformed, and scaled peak area. The additions of the compounds mentioned in the bottom of each panel were made from top to bottom. S succinate, 5 mM (striped grey); G glutamate, 5 mM; M malate, 5 mM; P pyruvate, 5 mM; Rot (blue) rotenone, 1 μM; Atpn (green) atpenin A5, 1 μM; Stigm (orange) 0.5 μM: stigmatellin; CN (yellow) cyanide, 1 mM. Data pooled from 3 to 37 independent experiments. * p < 0.05. (I) A cartoon representing the experimental timeline and extraction points (E.P.). Experiments always started the same as previously described, except 2.5 mM unlabeled glutamate was present and 2.5 mM [U-¹³C]-labelled glutamate was added just before anoxia when [O₂] was ~ 10 μM. Just prior to extraction, 500 μM N-ethylmaleimide, 1 μM rotenone, 1 μM atpenin, 1 μM myxothiazol, 1 mM arsenite, 100 μM p-hydroxymercurybenzoate, 100 μM aminooxyacetate and 1 mM cyanide were added to the suspension in the closed chamber. Samples were extracted as described in the methods section. (J) A model for pathways leading to- and from succinate. Note the directionality of the arrows: ΔS_M is positive in the fumarate to succinate direction and ΔS_T is positive in the export direction. Because no succinate was added externally, it is reasonable to assume that succinate exits through the IMM. (K–M) Average rates of succinate formation and transport expressed as velocity (ν). Rates are represented instead of absolute amounts to make different timeframes comparable: (K) 0–600 s; (L) 0–200 s; (M) 200–600 s. Magenta signifies succinate from glutamate, cyan from malate, green from transport across IMM. For (K–M) Combined standard uncertainty was calculated using the Gauss’ law of error propagation using linear approximation using the NIST uncertainty machine v1.5. Subsequently, covariance between succinate isotopologue amount fractions and between 2-oxoglutarate isotopologue amount fractions were taken into account, although they were negligible. No other consistent correlation was found among the uncertainties.