Before phosphorylation of ADP to ATP occurs at complex V, secondary to matrix proton uptake, respiration must supply the protons that are pumped out across the inner mitochondrial membrane to generate the proton motive force. As this is associated with electron transfer from one mobile electron carrier to another, it is essential that an electron is captured first by the next charge carrier before highly electronegative atoms such as O2 can capture the electrons that could result in an excess of superoxide radicals. Normally, this is kept to a minimum by several factors that keep forward electron transfer at a fast pace and inhibit reverse electron transfer. This is aided by tight distance coupling of the mobile carriers, the presence of hydrophobic amino acids in the respiratory complexes that keep water away from the electrons, and by the presence of mitochondrial and cytosolic superoxide dismutases, which scavenge for superoxide ions and convert them to hydrogen peroxide, a gas that can transverse membranes. Meanwhile, the reuptake of protons drives ATP synthesis, and the fewer protons entering the matrix for the ATP generated, the higher is the coupling ratio (ATP generated/protons taken up), a marker of mitochondrial efficiency. Proton leak is the matrix entry of H+ ions by any mechanism, for example via cation exchangers, other than via ATP synthase. Proton leak stimulates respiration because it decreases the energy of two components of the proton motive force, the voltage (ΔΨm), and proton (ΔpHm) gradients. Low levels of electron leak can lead to formation of hydrogen peroxide, adapted to use as a gaseous modulator of mitochondrial and cell function. Glycolytic and tricarboxylic acid cycle enzyme activities are regulated allosterically by substrates, metabolic intermediates, metal and organic cofactors, ATP/ADP ratios, and others. Respiration is modulated by the proton motive force and by factors that modulate it, like protons, but also by Ca2+ and K+ ions. The individual respiratory complexes can also be inhibited or stimulated; for example, complex IV is inhibited by nitric oxide or ATP. An understanding of these mechanisms typically requires dynamic measurements of multiple mitochondrial factors in situ or in vivo, such as membrane potential, pH gradient, redox state, respiration (O2 consumption), ATP/ADP ratios, enzyme activities, cation concentrations, and reactive oxygen species.