Oxidative phosphorylation can occur only in the presence of adequate supplies of its substrates. It is controlled not by allosteric mechanisms, but simply by substrate availability. Those substrates include ADP, Pi, O2, and an oxidizable metabolite that can generate reduced electron carriers - NADH and/or FADH2. Under different metabolic conditions any one of these four substrates can limit the rate of oxidative phosphorylation.

The dependence of oxidative phosphorylation on ADP reveals an important general feature of this process: Respiration is tightly coupled to the synthesis of ATP. Not only is ATP synthesis absolutely dependent on continued electron flow from substrates to oxygen, but electron flow in normal mitochondria occurs only when ATP is being synthesized as well. This regulatory phenomenon, called respiratory control, makes biological sense, because it ensures that substrates will not be oxidized wastefully. Instead, their utilization is controlled by the physiological need for ATP.

In most aerobic cells the level of ATP exceeds that of ADP by 4- to 10-fold. Respiration depends on ADP as a substrate for phosphorylation. When ATP is consumed at high rates, accumulation of ADP stimulates respiration, with concomitant activation of ATP resynthesis. Conversely, in a relaxed and well-nourished cell, ATP accumulates at the expense of ADP, and the depletion of ADP limits the rate of both electron transport and its own phosphorylation to ATP. Thus, the energy-generating capacity of the cell is closely attuned to its energy demands.

Experimentally, respiratory control is demonstrated by following oxygen utilization in isolated mitochondria (Figure 15.22). In the absence of added substrate or ADP, oxygen uptake, caused by oxidation of endogenous substrates, is slow. Addition of an oxidizable substrate, such as glutamate or malate, has but a small effect on the respiration rate. If ADP is then added, however, oxygen uptake proceeds at an enhanced rate until all of the added ADP has been converted to ATP, and then oxygen uptake returns to the basal rate. This stimulation of respiration is stoichiometric; that is, addition of twice as much ADP causes twice the amount of oxygen uptake at the enhanced rate. If excess ADP is present instead of oxidizable substrate, the addition of substrate in limiting amounts will stimulate oxygen uptake until the substrate is exhausted.

Maintenance of respiratory control depends on the structural integrity of the mitochondrion. Disruption of the organelle causes electron transport to become uncoupled from ATP synthesis. Under these conditions, oxygen uptake proceeds at high rates even in the absence of added ADP. ATP synthesis is inhibited, even though electrons are being passed along the respiratory chain and used to reduce O2 to water.

Uncoupling of respiration from phosphorylation can also be achieved chemically. Chemical uncouplers such as DNP or FCCP act by dissipating the proton gradient. Addition of an uncoupler to mitochondria stimulates oxygen utilization even in the absence of added ADP. No phosphorylation occurs under these conditions because there is no ADP to be phosphorylated.