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The carbon sequence is oxaloacetate $(4)$ plus an acetyl group $(2)$ to citrate $(6)$, then isocitrate $(6)$, alpha-ketoglutarate $(5)$, succinyl-CoA $(4)$, succinate $(4)$, fumarate $(4)$, malate $(4)$ and back to oxaloacetate $(4)$. The numbers count the cycling carbon skeleton, excluding coenzyme A. Two carbon dioxide releases accompany the changes $6\to 5\to 4$.^[ref]

Per acetyl group: three NADH, one FADH2 equivalent, one GTP or ATP by substrate-level phosphorylation, and two carbon dioxide. Succinate dehydrogenase retains FADH2 and transfers its Electrons to ubiquinone, forming ubiquinol. The FADH2 equivalent and ubiquinol count the same Electron pair.^[ref]

A useful check adds the stepwise changes. The carbon count is $4+2-1-1=4$; the NADH count is $1+1+1=3$. Each NADH carries two Electrons, and the succinate dehydrogenase route supplies another pair, so one turn exports $3\times 2+2=8$ Electrons. This arithmetic is an accounting identity, not an ATP yield calculation. Converting it to ATP requires the separate energy coupling and transport assumptions discussed under oxidative phosphorylation.

For a chosen ideal conversion of $5\mu \mathrm{mol}$ of acetyl groups, multiplication gives $15\mu \mathrm{mol}$ of NADH, $5\mu \mathrm{mol}$ of ubiquinol, $5\mu \mathrm{mol}$ of GTP and $10\mu \mathrm{mol}$ of carbon dioxide. The Electron output is $40\mu \mathrm{mol}$. If all these Electrons subsequently reach oxygen with no competing sinks, four Electrons per oxygen molecule require $10\mu \mathrm{mol}$ of oxygen. That is consumption by the connected electron transport chain; it is not an oxygen term in the cycle's own substrate ledger.

No cycle step directly consumes molecular oxygen. In ordinary aerobic mitochondrial operation, however, the mitochondrial respiratory chain must reoxidize NADH and ubiquinol to restore the oxidized electron carriers. Without that outlet, a finite oxidized-carrier pool cannot sustain continued turnover. Other electron acceptors and other forms of metabolism require their own analysis.^[ref]

The pyruvate dehydrogenase complex acts upstream: one three-carbon pyruvate gives one two-carbon acetyl group, one carbon dioxide and one NADH. These are additional to the cycle ledger.^[ref] Consequently, processing one pyruvate through both stages gives three carbon dioxide and four NADH, while the succinate dehydrogenase contribution and the substrate-level phosphorylation contribution each remain one. Counting the upstream step inside the cycle would obscure where the extra carbon dioxide and NADH arise.

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.

Malate is the anion obtained by removing both carboxyl protons from malic acid. Its protonation state distinguishes it from the acid.