ATP in Mitochondria: Quantitative Measurement, Regulation, and Physiological Role

Oxidative phosphorylation in mitochondria is the main source of ATP in most eukaryotic cells. Concentrations of ATP, ADP, and AMP affect numerous cellular processes, including macromolecule biosynthesis, cell division, motor protein activity, ion homeostasis, and metabolic regulation. Variations in ATP levels also influence concentration of free Mg²⁺, thereby extending the range of affected reactions. In the cytosol, adenine nucleotide concentrations are relatively constant and typically are around 5 mM ATP, 0.5 mM ADP, and 0.05 mM AMP. These concentrations are mutually constrained by adenylate kinases operating in the cytosol and intermembrane space and are further linked to mitochondrial ATP and ADP pools via the adenine nucleotide translocator. Quantitative data on absolute adenine nucleotide concentrations in the mitochondrial matrix are limited. Total adenine nucleotide concentration lies in the millimolar range, but the matrix ATP/ADP ratio is consistently lower than the cytosolic ratio. Estimates of nucleotide fractions show substantial variability (ATP 20-75%, ADP 20-70%, AMP 3-60%), depending on the organism and experimental conditions. These observations suggest that the 'state 4' - inhibition of oxidative phosphorylation in the resting cells due to the low matrix ADP and elevated proton motive force that impedes respiratory chain activity - is highly unlikely in vivo. In this review, we discuss proteins regulating ATP levels in mitochondria and cytosol, consider experimental estimates of adenine nucleotide concentrations across a range of biological systems, and examine the methods used for their quantification, with particular emphasis on the genetically encoded fluorescent ATP sensors such as ATeam, QUEEN, and MaLion.