ATP is synthesized in eukaryotic cells:

           1) in the cytosol by glycolysis and
           2) in mitochondria by oxidative phosphorylation.

In most animal cells more than 90 % of ATP is synthesised in mitochondria. However, in transgenic mice lacking cytochrome c fetal development could occur for two weeks (half of the pregnancy) without respiration and oxidative phosphorylation [27]. Cells in culture can indeed grow without oxidative phosphorylation [28]. It is suggested that during early fetal development respiration and oxidative phosphorylation are suppressed in order to prevent generation of ROS (reactive oxygen species) in the respiratory chain (see below).
 

"Respiratory control" = control of mitochondrial respiration by the availability of ADP. Respiratory control adapts the rate of respiration to the utilization of ATP.
 
First mechanism of respiratory control
 
  The control of mitochondrial respiration according to the utilization of ATP is generally explained by the chemiosmotic hypothesis (Mitchellian mechanism), based on the inhibition of proton pumps at high proton motive force delta p (mainly delta psi = 140-180 mV) [16]. ADP stimulates the ATP synthase (driven by delta psi), resulting in decreased delta psi and stimulation of proton pumps (see Fig. 5).
 
Second mechanism of respiratory control
 

 

 

 The second mechanism of respiratory control is based on inhibition of phosphorylated COX at high intramitochondrial ATP/ADP-ratios, leading to sigmoidal inhibition kinetics (Fig. 4) [193, 202]. ADP decreases the ATP/ADP-ratio in mitochondria, abolishes allosteric ATP-inhibition of COX, thus stimulates respiration. In isolated mitochondria COX is not rate-limiting [35,36], probably due to dephosphorylation of COX. The allosteric ATP-inhibition converts COX into the rate-limiting step of the respiratory chain, as verified by metabolic control analysis in cultured cells [32-34].
 

  In isolated mitochondria [16, 30] and with reconstituted COX [140, 161] a high proton motive force (mainly delta psi) is usually measured (150-200 mV). In contrast, low delta psi-values were determined in perfused hearts (100-150 mV) [31].
 
    Since the rate of ATP synthesis by the ATP synthase is maximal at low delta psi (100-120 mV) [29], the feedback inhibition of COX by high intramitochondrial ATP/ADP-ratios stabilizes low delta psi values (= 100-140 mV). Dephosphorylation of COX switches off the second mechanism of respiratory control (allosteric ATP-inhibition of COX) and increases delta psi, because proton pumps are then inhibited only at high delta psi values (150-200 mV) [16] (Fig. 5).


  
 

Fig. 5: Suggested control of cell respiration by the proton motive force
 (first mechanism of respiratory control) at high delta psi (140-180 mV), and by the intramitochondrial ATP/ADP-ratio (second mechanism of respiratory control) at low delta psi (100-140 mV) (taken from 214).

We propose that in vivo "stress hormones" which increase [Ca2+], increase delta psi via calcium-activated dephosphorylation of COX. The increase of delta psi as a consequence of increased [Ca2+] has in fact been measured in cultivated hepatocytes after addition of vasopressin or thapsigargin [37, 38].
 

 
ROS (reactive oxygen species) generation in mitochondria increases exponentially with increasing delta psi above 140 mV [39, 40, 41]. The production of ROS is assumed to occur in the respiratory chain by one-electron transfer from ubisemiquinone to dioxygen [42]. The second mechanism of respiratory control is independent of delta p [201] and keeps delta psi low, thus suppressing ROS generation, as illustrated in Fig. 5.

  
 



Fig. 6. Postulated control of delta psi and ROS formation in mitochondria (taken from 221). The figure presents schematically the inhibition of respiration at high
delta psi via the first mechanism of respiratory control [16] (red line), the inhibition of respiration by high ATP/ADP ratios via the second mechanism of respiratory control (allosteric ATP-inhibition, green line) [202], based on the membrane potential dependence of the ATP synthase [29] (blue line), and the delta psi dependence of ROS formation [39-41] (lila line). The suggested physiological range of delta psi is underlayed in light green.

 
 



An increased generation of ROS as the result of increasing cellular [Ca2+] was demonstrated in cultivated neurons after addition of glutamate [43] or N-methyl-D-aspartate [44].

Free radicals were measured by ESR in skeletal muscle samples from human, rat and mouse after excessive contractile activity, indicating increased ROS generation [45]. Muscle damage after excessive muscle activity was postulated to be based on the increased formation of free radicals.