 Solution

Titrate the omitted activities using small perturbations around the same steady-state condition, measuring oxygen consumption and calculating local slopes rather than assigning control from complete inhibition. For example, suitable ATP synthase, succinate dehydrogenase and carrier perturbations could test their contributions, with independent checks of inhibitor selectivity and activity loss. Measure ATP production alongside oxygen consumption, as well as mitochondrial membrane potential, to separate a change in phosphorylation efficiency from a change in electron flux. Check chamber oxygen availability, mitochondrial integrity, substrate abundance and assay reproducibility.

Repeat the control measurements across ADP demands and with defined alternative substrates. Carefully titrated FCCP can test respiratory capacity when proton-gradient backpressure is relieved, but the uncoupled state is a different operating condition and does not itself reveal the coupled-state coefficients. Fit the measured local sensitivities with uncertainty and test whether the expanded set satisfies the flux summation theorem. This distinguishes omitted control from technical inconsistency and shows whether the distribution changes with demand.