Physiological uncoupling of mitochondrial oxidative phosphorylation. Studies in different yeast species

Under non-phosphorylating conditions a high proton transmembrane gradient inhibits the rate of oxygen consumption mediated by the mitochondrial respiratory chain (state IV). Slow electron transit leads to production of reactive oxygen species (ROS) capable of participating in deleterious side reactions. In order to avoid overproducing ROS, mitochondria maintain a high rate of O₂ consumption by activating different exquisitely controlled uncoupling pathways. Different yeast species possess one or more uncoupling systems that work through one of two possible mechanisms: i) Proton sinks and ii) Non-pumping redox enzymes. Proton sinks are exemplified by mitochondrial unspecific channels (MUC) and by uncoupling proteins (UCP). Saccharomyces. cerevisiae and Debaryomyces hansenii express highly regulated MUCs. Also, a UCP was described in Yarrowia lipolytica which promotes uncoupled O₂ consumption. Non-pumping alternative oxido-reductases may substitute for a pump, as in S. cerevisiae or may coexist with a complete set of pumps as in the branched respiratory chains from Y. lipolytica or D. hansenii. In addition, pumps may suffer intrinsic uncoupling (slipping). Promising models for study are unicellular parasites which can turn off their aerobic metabolism completely. The variety of energy dissipating systems in eukaryote species is probably designed to control ROS production in the different environments where each species lives.     

Control of mitochondrial oxidative phosphorylation

The objective of this investigation is to analyze the two following problems of the regulation of mitochondrial oxidative phosphorylation: what is the extramitochondrial parameter that controls ATP production according to the cytoplasmic demands and how the control is distributed between various mitochondrial enzymes. On the basis of the data of Groen et al. (1982) it is shown that as the respiration rates ranged over 30-50% of the maximum (i.e. within the physiological region) the contribution of the adenine nucleotide translocator to the control of the ATP flux is no less than 90%, referring to the total contribution of all mitochondrial enzymes as 100%. Founding on the key role of the adenine nucleotide translocator it has been concluded that besides the extramitochondrial [ATP]/[ADP] ratio the absolute ADP concentration is another extramitochondrial signal controlling significantly the rate of oxidative phosphorylation.     

Mechanistic stoichiometry of mitochondrial oxidative phosphorylation

P/O ratios of rat liver mitochondria were measured with particular attention to systematic errors. Corrections for energy loss during oxidative phosphorylation were made by measurement of respiration as a function of mitochondrial membrane potential. The corrected values were close to 1, 0.5, and 1 at the three coupling sites, respectively. These values are consistent with recent measurements of mitochondrial proton transport.     

Uncouplers of rat-liver mitochondrial oxidative phosphorylation

1. The ability of a series of compounds to uncouple oxidative phosphorylation of rat-liver mitochondria has been investigated. 2. The compounds were: 2-amino-1,1,3-tricyanopropene; carbonyl cyanide phenylhydrazone and its m-chloro and p-trifluoromethoxy derivatives; 4,5,6,7-tetrachloro-, 5-chloro-4-nitro-, 5-nitro-and 4,5,6,7-tetrachloro-1-methyl-benzotriazole; 4-hydroxy-3,5-di-iodo-, 3,5-di-bromo-4-hydroxy- and 3,5-dichloro-4-hydroxy-benzonitrile; and pentafluorophenol. 3. In a medium the components and physical condition of which were, as far as possible, kept constant, each compound was tested for ability to stimulate adenosine triphosphatase, to stimulate respiration in the presence of pyruvate as substrate, to inhibit phosphate uptake and to prevent swelling by trimethyltin. 4. Each compound was also examined with respect to its ability to produce rapid rigor mortis in mice. 5. The biological properties were compared with the dissociation constant and the hexane–water partition coefficient for each compound. 6. With the exception of 4,5,6,7-tetrachloro-1-methylbenzotriazole, all the compounds behaved qualitatively as 2,4-dinitrophenol. 7. Within each class of compound there is a relation between biological activity and the physical attributes measured. 8. The most efficient uncouplers were the most acidic and the most hydrophobic.     

Flow-force relationships in mitochondrial oxidative phosphorylation

The rates of oxidation and phosphorylation in isolated rat-liver mitochondria have a steep dependence on the protonmotive force (delta mu H+) across the membrane. These experimentally observed relationships proved to be independent of the way in which delta mu H+ was varied. These results were obtained when the membrane potential (delta psi) was calculated from the distribution of K+ (in the presence of valinomycin). When triphenylmethylphosphonium (TPMP+) was used as a probe for delta psi, slightly different flow-force relationships were obtained. We conclude that unique relationships exist between delta mu H+ and the rates of oxidation and phosphorylation, and that under some conditions the behaviour of the probe TPMP+ is anomalous.     

Uncoupling of mitochondrial oxidative phosphorylation by hexetidine

To gain further insight into the biochemical properties of the antibacterial hexetidine, isolated rat liver mitochondria were added with this drug and investigation made of certain features related to mitochondrial bioenergetics. Hexetidine was found to cause oxidation of intramitochondrial pyridine nucleotides and stimulate the rate of oxygen uptake caused by respiratory substrates involving three, two and one site(s) of phosphorylation. Reversal of oxygen uptake inhibition by oligomycin was also determined. By investigating hexetidine effect on oxidative phosphorylation, hexetidine was found both to inhibit the rate of ATP synthesis and to cause ATP hydrolysis. Likewise, hexetidine capability to produce acidification of extramitochondrial medium and to collapse delta psi was also observed. The reported findings show that hexetidine exhibits uncoupling properties.     

A novel property of mitochondrial oxidative phosphorylation

DNA synthesis in isolated HeLa nuclei was measured by ³H-TTP incorporation in the presence of cytosol from S-phase cells. The addition of total calf thymus histone at low concentrations stimulated incorporation. Higher levels of added histone markedly inhibited DNA synthesis. The effects of added histone were dependent on the physiological state of the cells from which nuclei were isolated.     

Effect of parthenin on mitochondrial oxidative phosphorylation

The sesquiterpene lactone, 'parthenin' the toxic principle of the allergenic weed Parthenium hysterophorus, inhibited 'state 3' respiration and stimulated 'state 4' respiration in rat liver and kidney mitochondria as well as ATPase activity in the presence of Mg2+ ions. These properties indicate that the toxic action of parthenin may be related to its interference with oxidative phosphorylation.     

Inhibition of mitochondrial oxidative phosphorylation by adriamycin

The antitumour antibiotic, adriamycin, inhibited oxidative phosphorylation in freshly prepared mitochondria from the heart, liver and kidney of the rat. It abolished respiratory control and stimulated ATPase activity. Succinate oxidation by heart mitochondria was extremely sensitive to the drug when hexokinase was present in the reaction medium. The sensitive site has been identified to lie in the region between the succinate dehydrogenase flavoprotein and ubiquinone of the respiratory chain.     

A coulombic hypothesis of mitochondrial oxidative phosphorylation

A coulombic hypothesis of mitochondrial oxidative phosphorylation is presented, founded upon the evidence for negative fixed charge formation during electron transport chain activity. The intermediary force is electrostatic (psi H) and not electrochemical (delta mu H). The electrochemical potential of the chemiosmotic hypothesis is identified as a "phantom" parameter which owes its delusive existence to the procedures by which it is measured. The connection between psi H and the conditional delta mu H values is examined; it entails the use of a variable conversion factor, f, where delta mu H (mV) = f psi H, and the concept of the "protonic status" of the diffuse double layer. A number of problems which beset the chemiosmotic view are reappraised in the light of the new interpretation, and find authentic solutions.     

Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation

Dimerization or oligomerization of ATP synthase has been proposed to play an important role for mitochondrial cristae formation and to be involved in regulating ATP synthase activity. We found comparable oligomycin-sensitive ATPase activity for monomeric and oligomeric ATP synthase suggesting that oligomerization/monomerization dynamics are not directly involved in regulating ATP synthase activity. Binding of the natural IF1 inhibitor protein has been shown to induce dimerization of F1-subcomplexes. This suggested that binding of IF1 might also dimerize holo ATP synthase, and possibly link dimerization and inhibition. Analyzing mitochondria of human rho zero cells that contain mitochondria but lack mitochondrial DNA, we identified three subcomplexes of ATP synthase: (i) F1 catalytic domain, (ii) F1-domain with bound IF1, and (iii) F1-c subcomplex with bound IF1 and a ring of subunits c. Since both IF1 containing subcomplexes were present in monomeric state and exhibited considerably reduced ATPase activity as compared to the third subcomplex lacking IF1, we postulate that inhibition and induction of dimerization of F1-subcomplexes by IF1 are independent events. F1-subcomplexes were also found in mitochondria of patients with specific mitochondrial disorders, and turned out to be useful for the clinical differentiation between various types of mitochondrial biosynthesis disorders. Supramolecular associations of respiratory complexes, the "respirasomes", seem not to be the largest assemblies in the structural organization of the respiratory chain, as suggested by differential solubilization of mitochondria and electron microscopic analyses of whole mitochondria. We present a model for a higher supramolecular association of respirasomes into a "respiratory string".     

Slip and leak in mitochondrial oxidative phosphorylation

During oxidative phosphorylation by mammalian mitochondria part of the free energy stored in reduced substrates is dissipated and energy is released as heat. Here I review the mechanisms and the physiological significance of this phenomenon.     

P/O ratios of mitochondrial oxidative phosphorylation

Mitochondrial mechanistic P/O ratios are still in question. The major studies since 1937 are summarized and various systematic errors are discussed. Values of about 2.5 with NADH-linked substrates and 1.5 with succinate are consistent with most reports after apparent contradictions are explained. Variability of coupling may occur under some conditions but is generally not significant. The fractional values result from the coupling ratios of proton transport. An additional revision of P/O ratios may be required because of a report of the structure of ATP synthase (D. Stock, A.G.W. Leslie, J.E. Walker, Science 286 (1999) 1700-1705) which suggests that the H+/ATP ratio is 10/3, rather than 3, consistent with P/O ratios of 2.3 with NADH and 1.4 with succinate, values that are also possible.     

Influence of anion flux upon mitochondrial oxidative phosphorylation

We have measured mitochondrial ATP synthesis during passive anion influx and find that influx of phosphate leads to diminished efficiency (as reflected in the ATP:0 ratio) whereas influx of acetate produced enhanced efficiency. The anions, sulfate, propionate, and thiocyanate, are without influence on the ATP:0 ratio. It is likely that the opposite effects of phosphate and acetate on the ATP:0 ratio reflect phosphate-acetate exchange, and that acetate influx produces its positive effect on ATP synthesis by promoting phosphate efflux. Thus, phosphate efflux may be associated with increased, and phosphate influx, with decreased energy conservation.     

Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

Supramolecular structure of the mitochondrial oxidative phosphorylation system

The protein complexes of the mitochondrial oxidative phosphorylation system were recently reported to form supramolecular assemblies termed respiratory supercomplexes or respirasomes. These supercomplexes are considered to be of great functional importance. Here we review new insights into supercomplex structure and physiology.     

Local energized proton hypotheses of mitochondrial oxidative phosphorylation

Two hypotheses are compared each interpreting mitochondrial energy transduction in terms of a localized form of proton activity. Their differences are seen to be profound and far-reaching. It is concluded that the "chemiosmotic/local energized proton dialogue" as conducted hitherto has offered a very incomplete and restricted analysis of the problems of mitochondrial oxidative phosphorylation.