(A) Schematic depicting the function of each RC component and the corresponding sites of inhibition for piericidin, antimycin, and oligomycin. Complexes I–IV transfer high- energy reducing equivalents from NADH and FADH2 to O2, generating a proton gradient in the process. Complex V utilizes this gradient to synthesize ATP. CoQ, coenzyme Q; CytC, cytochrome C.
(B) Heat map representing changes in metabolite concentrations upon inhibition of Complex I, III, or V, as assessed by whole-cell and mitochondrial metabolomics. For each metabolite and inhibitor, the mean log2-transformed fold change is relative to the corresponding whole-cell or matrix concentration of vehicle-treated cells (n = 3). To be included in the heat map, metabolites had to change at least 2-fold upon inhibition of an RC complex. See Table S2 for additional criteria used to generate this heat map and for the concentrations of all metabolites.
(C) Whole-cell and matrix profiles during RC dysfunction are substantially different. Principal component analysis of metabolite changes in Figure 3B as assessed by profiling of the mitochondrial matrix (blue) or whole-cells (black).
(D) RC inhibition lowers matrix PEP.
(E) RC inhibition increases matrix saccharopine.
(F) The NADH/NAD imbalance during RC dysfunction is more pronounced in the matrix than on the whole-cell level.
(G) The relationship between matrix aspartate and the matrix NADH/NAD ratio can be modeled as a power function. Log10-transformed values of matrix aspartate concentrations (units of M) and NADH/NAD ratios were compared across different states of RC function and a Pearson correlation coefficient was calculated.
(H) Inhibition of Complexes I and III increases matrix GSH/GSSG ratios. For all panels, unless indicated otherwise, all experiments were performed in DMEM without pyruvate and all measurements are normalized to the corresponding whole-cell or matrix concentrations of vehicle-treated cells (mean ± SEM, n = 3, *p < 0.05).