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- Baglivo 2023 ESCI Prague + (Cytochrome c is the redox link between Com … Cytochrome c is the redox link between Complexes CIII and CIV of the mitochondrial electron transfer system. Cytochrome c release across the mitochondrial outer membrane (mtOM) is associated with apoptosis. Stimulation of respiration by added cytochrome c indicates damage of the mtOM. This respirometric test for mtOM integrity provides a quality control to exclude potential injuries caused experimentally during mitochondrial preparation by tissue homogenization or plasma membrane permeabilization.</br></br>Selecting the step of cytochrome c addition in substrate-uncoupler-inhibitor-titration (SUIT) protocols is of primary importance for diagnostic analysis. This study presents a quality control of SUIT protocols using the cytochrome c test.</br></br>We applied SUIT protocols RP1 and RP2 to address the cytochrome c effect in different respiratory states. The first steps in RP1 use pyruvate & malate to analyze coupling control in the NADH-pathway. RP2 focuses on fatty acid oxidation (FAO), considering malate-linked anaplerosis. We tested these protocols in HEK 293T permeabilized cells and beef liver and heart homogenate. </br></br>Cytochrome c control efficiency [1] was correlated when cytochrome c was added in the ADP-activated OXPHOS state of the NADH-pathway and in FAO. Following 0.1 mM malate in RP2, addition of cytochrome c before or after titration of fatty acid did not affect the O<sub>2</sub> flux in FAO. Optimization of digitonin concentrations for plasma membrane permeabilization of cultured cells was essential to avoid damage of the mtOM. Conclusions: Underestimating respiratory capacities in mitochondrial preparations is avoided by adding cytochrome c as an early step of SUIT protocols. However, cytochrome c added in the LEAK state causes an unexplained activation of respiration.EAK state causes an unexplained activation of respiration.)
