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An electron carrier cycles between oxidation and reduction. Ubiquinone carries two Electrons, while cytochrome c carries one at a time.^[ref]

For example, transferring the two Electrons supplied by one NADH requires two one-Electron deliveries by cytochrome c. It does not require two permanently consumed cytochrome c molecules: an available electron carrier can unload, return and reload. Electron carriers recycle material while the net redox conversion supplies free energy.

Respiratory control can be investigated outside an intact biological cell by supplying isolated mitochondria with substrates and ADP. This makes the boundary between oxygen consumption and ATP formation experimentally accessible. The historical mitochondrial oxygen electrode assay describes the 1955 experiment; the following authored protocol adds modern preparation and inhibitor controls.

In Functional Assessment of Isolated Mitochondria In Vitro (2009), Ian R. Lanza and K. Sreekumaran Nair describe human skeletal-muscle biopsy mincing, cold protease treatment, homogenization and differential centrifugation. A low-speed spin removes coarse material; subsequent spins collect and wash mitochondria. A stirred, closed $2\mathrm{mL}$ Oxygraph-2k chamber at $37{}^{\circ }\mathrm{C}$ uses a calibrated polarographic oxygen electrode. Its signal gives dissolved oxygen concentration; the negative time derivative gives consumption. Instrument corrections include electrode consumption and oxygen leakage.^[ref]

Their additions include glutamate plus malate, an ADP pulse, then saturating ADP. Added cytochrome c tests outer-membrane damage: stimulation suggests loss of endogenous cytochrome c. Later oligomycin inhibits ATP synthase; FCCP produces respiratory uncoupling; antimycin A inhibits respiratory complex III to estimate residual background. Figure 6 includes intervening succinate dehydrogenase and respiratory complex I interventions; the modeled traces below simplify that sequence. Isolation can damage or select mitochondria, and saturating assay conditions differ from those in living biological tissue.^[ref]

The control logic matters more than a single steep slope. If ADP accelerates consumption and a finite pulse is followed by slowing, demand is affecting respiratory flux. If oligomycin slows consumption, but subsequent FCCP accelerates it, the electron transport chain can still turn over while ATP synthase remains inhibited. That contrast separates restricted coupled turnover from a completely inactive chain. Failure of FCCP to restore a rate is less specific: substrate limitation, damage, low oxygen or excessive uncoupler can also restrict respiration.

Neither the cytochrome c test alone nor a large respiratory control ratio establishes every aspect of mitochondrial integrity. Likewise, oxygen consumption is not a direct ATP assay: respiratory uncoupling provides a concrete counterexample. The preparation, additions and normalization denominator determine what comparison the experiment can support.

NADH supplies two Electrons to respiratory complex I, then ubiquinone, respiratory complex III, cytochrome c and cytochrome c oxidase; terminal oxygen reduction forms water. Succinate dehydrogenase, or respiratory complex II, supplies another entry to ubiquinone. Respiratory complex I, respiratory complex III and cytochrome c oxidase move protons outward; respiratory complex II does not.^[ref]

For an original Electron balance, supply exactly $6\mu \mathrm{mol}$ of NADH to an ideal preparation with no other electron donor or competing electron acceptor. The donor supplies $12\mu \mathrm{mol}$ of Electrons. Complete reduction of one oxygen molecule needs four Electrons, so the corresponding oxygen consumption is $3\mu \mathrm{mol}$. Adding the two formal half-reactions makes the result explicit:The cancelling Electrons determine the donor-to-oxygen ratio. These chemical equations do not count transmembrane protons or determine an ATP yield. Chemical protons consumed in water formation and transported protons have different roles in the ledger.

The outer mitochondrial membrane separates the surrounding cytosol from the intermembrane space. Channels permit passage of many small molecules, while the inner mitochondrial membrane supplies the main selective barrier between them and the mitochondrial matrix.^[ref]

Small-solute access does not imply unrestricted access for proteins. The cytochrome c addition in the isolated mitochondria respiration experiment tests a consequence of damage to this boundary. Organelle protein import describes purposeful delivery across mitochondrial boundaries.