Biochemistry - The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

How electrons meet oxygen, how ATP is formed in the process, and some related phenomena
Electron Transport Chain and Oxidative Phosphorylation
Stoichiometry and Sites of Oxidative Phosphorylation

In vitro ATP Synthesis in tissue homogenates was first observed in 1937 by Kalckar, who wrote an engaging historical review on the subject [71]. A major milestone followed in 1941 when Ochoa carried out the first reliable measurement of the P/O ratio. The P/O ratio represents the number of ATP molecules formed per atom of oxygen consumed during Respiration. It is also equal to the number of ATP molecules generated when a pair of electrons is transferred down the Electron Transport Chain. Ochoa established that during the Oxidation of Pyruvate to acetyl-CoA and CO₂ (a process that feeds two electrons into the transport chain), the P/O ratio is approximately three. This value has been repeatedly confirmed ever since. However, it should be clearly understood that measuring the P/O ratio is fraught with experimental difficulties, which have led to numerous errors—some made even in recent times. One METHOD FOR DETERMINING the P/O ratio relies on the Quantitative determination of ATP described in the legend to Fig. 8-11.

The experimentally observed P/O ratio of ~3 for The oxidation of pyruvate and many other substrates that supply NADH to The electron transport chain indicates that there are three distinct sites of ATP formation along the chain. It was soon demonstrated that this ratio is only two for the oxidation of succinate. This led to the hypothesis that one of the phosphorylation sites (site I) is located between NADH and ubiquinone, prior to the convergence with the succinate branch.

In 1949, Lehninger used ascorbate and tetramethylphenylenediamine (TMPD, Table 10-3) to feed electrons into the chain at the level of cytochrome c. The ascorbate→TMPD→cytochrome c sequence operates independently of Enzymes. Later on, researchers successfully used cytochrome c directly as an electron donor. In each case, only a single ATP molecule was formed, which was entirely expected if only site III lies to the right of cytochrome c. Somewhat later, Lardy localized site I by using ferricyanide in the presence of antimycin A as an artificial oxidant for NADH oxidation. Under these conditions, a P/O ratio of one was also observed. Finally, in 1955, Slater demonstrated that the passage of electrons from succinate to cytochrome c likewise yields only one ATP molecule, which is generated at site II.