Coupled mitochondria

FIGURE 19-18 Coupling of electron transfer and ATP synthesis in mitochondria. In experiments to demonstrate coupling, mitochondria are suspended in a buffered medium and an 02 electrode monitors 02 consumption. At intervals, samples are removed and assayed for the presence of ATP. (a) Addition of ADP and P, alone results in little or no increase in either respiration (02 consumption black) or ATP synthesis (red). When succinate is added, respiration begins immediately and... 

Respiring, coupled mitochondria (which maintain a high transmembrane potential) release Ca2+ through two pathways that can be studied in isolated mitochondria after the addition of RR to block Ca2+ reuptake. 

Wodtke, E. (1974). Effects of acclimation temperature on the oxidative metabolism of the eel (Anguilla anguilla). 1. Liver and red muscle. Changes in the mitochondrial content and in the oxidative capacity of isolated coupled mitochondria. Journal of Comparative Physiology 91,309-332. 

The maximum pmf generated in State 4 has been measured by various groups using several techniques (see Refs. 8,26,27). The results vary between about 160 and 240 mV. However, from the observed shifts of the redox equilibria in the respiratory chain imposed by the pmf (or by ATP via the pmf), it can be concluded that the functionally relevant pmf must be at least 200 mV in State 4 of well-coupled mitochondria [8]. (This need not be equivalent with the pmf between the bulk aqueous C and M phases). This limits the H /e ratio in State 4 to maximal values of 5.6 and 3.8 for the spans from NADH and ubiquinol to oxygen, respectively [8]. These are indeed upper limit stoicheiometries, attainable, of course, only at thermodynamic equilibrium between respiration and proton translocation. The true values must therefore be lower, particularly as the terminal step in the chain, i.e., oxidation of cytochrome c by Oj is irreversible (see Refs. 28, 29 and below). 

An interesting hypermetabolic myopathy was discovered and biochemically explored by Luft et al. (L7). There was no evidence of hyperthyroidism, and mitochondria from biopsied muscle had a high rate of respiration and a loosely coupled state of oxidative phosphorylation. A few other cases of unusual myopathies with loosely coupled mitochondria have since been described (e.g., S12), although it does not seem that this is a single disease entity. 

Inhibition of the electron transport chain in coupled mitochondria can occur at any of the three constituent functional processes electron transport per se, formation of ATP, or antiport translocation of ADP/ATP (Table 16-1). The best known inhibitor of the ADP/ATP translocase is atractyloside in the presence of which no ADP for phosphorylation is transported across the inner membrane to the ATP synthase and no ATP is transported out. In the absence of ADP phosphorylation the proton gradient is not reduced allowing other protons to be extruded into the intermembrane space because of the elevated [H+], and thus electron transfer is halted. Likewise the antibiotic oligomycin directly inhibits the ATP synthase, causing a cessation of ATP formation, buildup of protons in the intermembrane space, and a halt in electron transfer. Similarly, a blockade of complex I, III, or IV that inhibits electron flow down the chain to would also stop both ATP formation and ADP/ATP translocation across the inner mitochondrial membrane. 

The direct anodic oxidation of cytochrome c at a bipyridyl-modified electrode has already been incorporated in enzyme electrodes for lactate, carbon monoxide, and hydrogen peroxide. Here, cytochrome c is reduced by cytochrome b2, CO oxidoreductase, or horseradish peroxidase and anodically reoxidized. Cytochrome c has also been applied to couple mitochondria and chloroplasts to redox electrodes (Albery et al. 1987). Although no practically applicable sensor has been constructed as yet, this principle offers a new avenue to the determination of inhibitors of photosynthesis or respiration (Cardosi and Turner, 1987). 

The addition of oxygen to the anaerobic coupled mitochondria by an oxygen pulse technique induces biphasic oxidation of type b cytochromes... 

Cytochrome 6-565 is only partially reduced by succinate in anaerobic uncoupled mitochondria and can be fully reduced by succinate in coupled mitochondria ( 2). At steady state this cytochrome is more reduced than other type 6 cytochromes and its reduction is further increased by energization of the mitochondrial membrane. Thus, cytochrome 6-565 is spectrally and functionally very similar to cytochrome 6t 16,18-20) in animal mitochondria. However, in contrast to cytochrome 6t, the En of this cytochrome is not affected by the energy state of mitochondria 22), and its a. band seems to be a single band with no appreciable shoulder even at 77°K 265). 

States" of mitochondria and spectrophoto-metric observation. Chance and Williams defined five states of tightly coupled mitochondria of these, states 3 and 4 are most often mentioned. If no oxidizable substrate or ADP is added the mitochondria have a very low rate of oxygen uptake and are in state I. If oxidizable substrate and ADP are added rapid O2 uptake is observed, the rate depending upon the rate of flow of electrons through the electron transport chain. This is state 3. As respiration occurs the coupled phosphorylation converts ADP into ATP, exhausting the ADP. Respiration slows to a very low value and the mitochondria are in state 4. If the substrate is present in excess, addition of more ADP will return the mitochondria to state 3. 

According to Holian et al. [53], however, respiration in tightly coupled mitochondria is controlled by the extramitochondrial ATP/ADP XP ratio (the phosphate potential) rather than by ATP/ADP. Since Pj is not involved in ADP transport it was concluded that ADP transport could not be rate-limiting. However, since under flux conditions both Pj and ADP have to enter to allow exit of ATP, it will be impossible to uncouple the effect of P, and adenine nucleotide transport. This... 

As discussed, ATP synfliesis is driven by flie proton motive force of the electrochemical gradient set up during the electron flow through the respiratory chain. For this reason, the two processes of oxidation and phosphorylation are described as being coupled (oxidative phosphorylation). In normally functioning, tightly coupled mitochondria oxidation proceeds primarily when ATP is synthesized from ADP. The dependence of respiration on ADP levels is defined as respiratory control, a key property of coupled mitochondria. The respiratory... 

Phospholipase A2 enzymes also have other important metabolic functions in addition to the overall destruction of phospholipids as catalysed by digestive pancreatic or venom enzymes. An enzyme in mitochondrial membranes seems to be intimately connected with the energy state of this organelle. Thus, the phospholipase is inactive in fully coupled mitochondria and only becomes active when ATP and respiratory control drop to low levels. Also, the widespread distribution of phospholipases A2 allows many tissues to perform retailoring of the molecular species of membrane lipids by the Lands mechanism. In this process, named after Lands, the American biochemist who first described it, cleavage of the acyl group from the sn-2 position yields a lysophospholipid which can be re-acylated with a new fatty acid from acyl-CoA (Figure 7.8). 

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