Mitochondrial function and redox state in mammalian embryos

Mitochondria play a central and multifaceted role in the mammalian egg and early embryo, contributing to many different aspects of early development. While the contribution of mitochondria to energy production is fundamental, other roles for mitochondria are starting to emerge. Mitochondria are central to intracellular redox metabolism as they produce reactive oxygen species (ROS, the mediators of oxidative stress) and they can generate TCA cycle intermediates and reducing equivalents that are used in antioxidant defence. A high cytosolic lactate dehydrogenase activity coupled with dynamic levels of cytosolic pyruvate is responsible for a very dynamic intracellular redox state in the oocyte and embryo. Mammalian embryos have a low glucose metabolism during the earliest stages of development, as both glycolysis and the pentose phosphate pathway are suppressed. The mitochondrial TCA cycle is therefore the major source of reducing equivalents in the cytosol so that any change in mitochondrial function in the embryo will be reflected in changes in the intracellular redox state. In the mouse, the metabolic substrates used by the oocyte and early embryo each have a different impact on the intracellular redox state. Pyruvate which oxidises the cytosolic redox state, acts as an energetic and redox substrate whereas lactate, which reduces the cytosolic redox state, acts only as a redox substrate. Mammalian early embryos are very sensitive to oxidative stress which can cause permanent developmental arrest before zygotic genome activation and apoptosis in the blastocyst. The oocyte stockpiles antioxidant defence for the early embryo to cope with exogenous and endogenous oxidant insults arising during early development. Mitochondria provide ATP for glutathione (GSH) production during oocyte maturation and also participate in the regeneration of NADPH and GSH during early development. Finally, a number of pathological conditions or environmental insults impair early development by altering mitochondrial function, illustrating the centrality of mitochondrial function in embryo development.     

The effects of phosphate and electron transport on the carbonyl cyanide m-chlorophenylhydrazone-induced ATPase of rat-liver mitochondria

1. The effects of phosphate and electron transport on the ATPase induced in ratliver mitochondria by the uncoupler carbonyl cyanide m-chlorophenylhydrazone have been measured at different uncoupler concentrations and compared with those of ATP, oligomycin and aurovertin.

2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase.

3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (> 0.2 μM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin.

4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations.

5. It is proposed that the steady-state concentration of endogenous P_i may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous P_i.     

Importance of the flux of phosphate across the inner membrane of kidney mitochondria for the activation of glutaminase and the transport of glutamine

The effect of mersalyl, an inhibitor of phosphate transport across the inner mitochondrial membrane, was investigated on the uncoupled respiration of pig kidney mitochondria in the presence of glutamine as substrate and on the activity of the phosphate-dependent glutaminase in the intact organelles. In addition, the submitochondrial location of the enzyme was reinvestigated.

1. (1) It was found that mersalyl completely inhibits uncoupled respiration of the mitochondria in the presence of glutamine as substrate, whereas respiration with glutamate was not affected. The same amount of mersalyl which inhibits coupled oxidation of glutamine also inhibits coupled oxidation of glutamate and some other substrates.

2. (2) Mersalyl strongly inhibited the activation of glutaminase in intact mitochondria only in the presence of inhibitors of electron transport or of an uncoupler. The addition of a detergent prevented or fully released the inhibition. The effect of mersalyl was observed even when the mitochondria were pre-incubated with phosphate or incubated in the phosphate-free medium. If mersalyl and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added 3 min after pre-incubation with phosphate the same intramitochondrial concentration of the anion as in control experiments was found, whereas the activity of glutaminase was severely inhibited. These findings suggest that the activation of the enzyme by phosphate in intact nonenergized mitochondria occurs only if the activator moves across the inner mitochondrial membrane.

3. (3) Mersalyl (plus CCCP) markedly decreased [¹⁴C]glutamine- and [³²P]-phosphate-permeable mitochondrial spaces. A close correlation between the decrease of phosphate and glutamine permeable spaces and the inhibition of glutaminase activity was found.

4. (4) If the activation energy of the enzyme was determined with frozen mitochondrial preparations, a discontinuity or break in the Arrhenius plot was observed, whereas the presence of a detergent completely abolished the break. Digitonin or ultrasonic treatment of the mitochondria followed by separation of the membrane and the soluble fraction revealed that glutaminase is a membrane-bound enzyme.

On the basis of these findings it is concluded that there is an association between the transport of phosphate on one side and the transport of glutamine and glutaminase activity on the other. It is possible that the movement of phosphate across the membrane activates the enzyme which facilitates diffusion of glutamine down a concentration gradient. However, the existence of a specific glutamine-phosphate carrier is not ruled out.     

Mitochondrial redox state as a potential detector of liver dysoxia in vivo

Dysoxia canbe defined as ATP flux decreasing in proportion toO₂ availability with preserved ATPdemand. Hepatic venous -hydroxybutyrate-to-acetoacetate ratio(-OHB/AcAc) estimates liver mitochondrial NADH/NAD and may detectthe onset of dysoxia. During partial dysoxia (as opposed to anoxia),however, flow may be adequate in some liver regions, diluting effluentfrom dysoxic regions, thereby rendering venous -OHB/AcAc unreliable.To address this concern, we estimated tissue ATP whilegradually reducing liver blood flow of swine to zero in a nuclearmagnetic resonance spectrometer. ATP flux decreasing withO₂ availability was taken asO₂ uptake(O₂) decreasing inproportion to O₂ delivery(O₂);and preserved ATP demand was taken as increasingP_i/ATP.O₂, tissueP_i/ATP, and venous -OHB/AcAcwere plotted againstO₂to identify critical inflection points. Tissue dysoxia required meanO₂for the group to be critical for bothO₂ and forP_i/ATP. CriticalO₂values for O₂ andP_i/ATP of 4.07 ± 1.07 and 2.39 ± 1.18 (SE) ml · 100 g¹ · min¹,respectively, were not statistically significantly different but notclearly the same, suggesting the possibility that dysoxia might havecommenced after O₂ begandecreasing, i.e., that there could have been"O₂ conformity." CriticalO₂for venous -OHB/AcAc was 2.44 ± 0.46 ml · 100 g¹ · min¹(P = NS), nearly the same as that forP_i/ATP, supporting venous -OHB/AcAc as a detector of dysoxia. All issues considered, tissue mitochondrial redox state seems to be an appropriate detector ofdysoxia in liver.

     

Mitochondrial carriers function as monomers

Mitochondrial carriers link biochemical pathways in the mitochondrial matrix and cytosol by transporting metabolites, inorganic ions, nucleotides and cofactors across the mitochondrial inner membrane. Uncoupling proteins that dissipate the proton electrochemical gradient also belong to this protein family. For almost 35 years the general consensus has been that mitochondrial carriers are dimeric in structure and function. This view was based on data from inhibitor binding studies, small-angle neutron scattering, electron microscopy, differential tagging/affinity chromatography, size-exclusion chromatography, analytical ultracentrifugation, native gel electrophoresis, cross-linking experiments, tandem-fusions, negative dominance studies and mutagenesis. However, the structural folds of the ADP/ATP carriers were found to be monomeric, lacking obvious dimerisation interfaces. Subsequently, the yeast ADP/ATP carrier was demonstrated to function as a monomer. Here, we revisit the data that have been published in support of a dimeric state of mitochondrial carriers. Our analysis shows that when critical factors are taken into account, the monomer is the only plausible functional form of mitochondrial carriers. We propose a transport model based on the monomer, in which access to a single substrate binding site is controlled by two flanking salt bridge networks, explaining uniport and strict exchange of substrates.     

Anomalies in the kinetics of photosynthetic oxygen emission in sequences of flashes revealed by matrix analysis. Effects of carbonyl cyanide m-chlorophenylhydrazone and variation in time parameters

The model of Kok et al. (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) is considered the best kinetic explanation of the damped oscillations of O₂ evolution induced in higher plants by a sequence of brief saturating flashes. Matrix analysis applied to this model shows that the parameters involved (distribution of S states at zero time, probabilities of transition between states induced by a flash) cannot be completely known from the O₂ yield sequence, Y_n. However, four quantities, with limited content of information, are readily derived from data, without additional assumptions. They are σ₁, σ₂ and σ₃, three quasi-symmetrical functions of the transition coefficients, and $\text{Y}$, a weighed average of four consecutive Y_n values. The extent of misses and double hits and their variations can be qualitatively ascertained by inspection of the relative values of σ₁, σ₂ and σ₃. In a regular sequence (strictly obeying Kok's model), all four quantities should be constant along the time axis.It is shown that actual sequences are seldom regular, in particular in the following conditions: (1) variable flashing frequency, (2) addition of carbonylcyanide m-chlorophenylhydrazone, (3) incomplete deactivation, (4) change of flashing frequency at steady state.In order to account for these anomalies, it is proposed to modify Kok's model by introducing, in parallel to the four state storage entity (S states), a side carrier C, which can reversibly exchange a positive charge with it. In the new model, the transition coefficients are essentially time varying, thus producing a nonregular behaviour of Y_n sequences.     

Mitochondrial redox systems as central hubs in plant metabolism and signaling

Plant mitochondria are indispensable for plant metabolism and are tightly integrated into cellular homeostasis. This review provides an update on the latest research concerning the organization and operation of plant mitochondrial redox systems, and how they affect cellular metabolism and signaling, plant development, and stress responses. New insights into the organization and operation of mitochondrial energy systems such as the tricarboxylic acid cycle and mitochondrial electron transport chain (mtETC) are discussed. The mtETC produces reactive oxygen and nitrogen species, which can act as signals or lead to cellular damage, and are thus efficiently removed by mitochondrial antioxidant systems, including Mn-superoxide dismutase, ascorbate–glutathione cycle, and thioredoxin-dependent peroxidases. Plant mitochondria are tightly connected with photosynthesis, photorespiration, and cytosolic metabolism, thereby providing redox-balancing. Mitochondrial proteins are targets of extensive post-translational modifications, but their functional significance and how they are added or removed remains unclear. To operate in sync with the whole cell, mitochondria can communicate their functional status via mitochondrial retrograde signaling to change nuclear gene expression, and several recent breakthroughs here are discussed. At a whole organism level, plant mitochondria thus play crucial roles from the first minutes after seed imbibition, supporting meristem activity, growth, and fertility, until senescence of darkened and aged tissue. Finally, plant mitochondria are tightly integrated with cellular and organismal responses to environmental challenges such as drought, salinity, heat, and submergence, but also threats posed by pathogens. Both the major recent advances and outstanding questions are reviewed, which may help future research efforts on plant mitochondria.

Plant mitochondria are key components of redox homeostasis and play vital roles in regulating cellular metabolism, thereby affecting development and stress tolerance at the whole plant level.

Advances

• Improved quantitative MS-based approaches have accelerated the study of mitochondrial protein abundance, turnover and PTMs.
• Mitochondrial enzymes and cellular compartments operate interactively and efficiently exchange substrates.
• Roles for mitochondrial retrograde signaling in plant growth, during physiologically relevant stress conditions and in interaction with other organelles such as the chloroplasts, have been clarified.
• Further insights into mitochondrial antioxidant and peroxidase systems and how they affect other redox systems, enzymes, and whole plant growth have been generated.
• Our understanding of how mitochondria help plants power development and cope with adversity has improved.

     

Coupling between redox and acid-base energy by cytochrome b-564 in Baker's yeast mitochondria

Baker's yeast mitochondrial cytochrome b-564 is characterized by exhibiting both a labile pH-independent high-potential form (E'o, pH 7 = + 190 mV) and a stable pH-dependent (pKa = 6.8) low-potential form (E'o, pH 7 = + 70 mV). The different behavior of these two forms of cytochrome b-564 versus pH seems to be a decisive factor for transduction of redox energy into acid-base energy in oxidative phosphorylation site 2. Deenergizing treatments, such as ADP plus Pi, result in the conversion of all the mitochondrial cytochrome b-564 into its low-potential form, whereas energization with ATP specifically transforms the cytochrome into its high-potential form, the ATP effect being neutralized by the ATPase inhibitor oligomycin and by the uncoupler FCCP. Accordingly, a minimal model for coupling between redox energy and acid-base energy through an electronically energized and protonated ferricytochrome b-564 intermediate is proposed. The energy-transducing properties of mitochondrial cytochrome b-564 seems to be shared by chloroplast cytochrome b-559.     

Mitochondrial compartmentalization of redox processes

Knowledge of location and intracellular subcompartmentalization is essential for the understanding of redox processes, because oxidants, owing to their reactive nature, must be generated close to the molecules modified in both signaling and damaging processes. Here we discuss known redox characteristics of various mitochondrial microenvironments. Points covered are the locations of mitochondrial oxidant generation, characteristics of antioxidant systems in various mitochondrial compartments, and diffusion characteristics of oxidants in mitochondria. We also review techniques used to measure redox state in mitochondrial subcompartments, antioxidants targeted to mitochondrial subcompartments, and methodological concerns that must be addressed when using these tools.     

Binding affinity and biological activity of oxygen and sulfur isosteres at melatonin receptors as a function of their hydrogen bonding capability

Analogues of melatonin (1) and of N-acetyl 5-ethoxytryptamine (3) in which the oxygen atoms are replaced by sulfur have been prepared and tested against human and amphibian melatonin receptors. All sulfur analogues show a decreased binding affinity at human MT1 and MT2 receptors and a reduced potency as melatonin agonists on the Xenopus melanophore assay. The 5-methoxy oxygen of melatonin is significantly more important for receptor binding than the amide oxygen. N-Acetyl 5-ethoxytryptamine shows a decrease in both binding affinity and potency in comparison with melatonin. In this series, replacing either the ethoxy or amide oxygen by sulfur has a similar but smaller effect on both binding affinity and potency. Using K(B)(H) values from Abraham's equations we have assessed the possibility of estimating EC50 values for sulfur isosteres from the EC50 values of their oxygen analogues.     

Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

Mitochondrial redox control of matrix metalloproteinases

Reactive oxygen species (ROS) are constantly generated in aerobic organisms during normal metabolism and in response to both internal and external stimuli. Imbalances in the production and removal of ROS have been hypothesized to play a causative role in numerous disease pathologies such as cancer, ischemia/reperfusion injury, and degenerative diseases such as photoaging, atherosclerosis, arthritis, and neurodegeneration. A feature often associated with these diseases is a malfunctioning of the connective tissue remodeling process due to increased activity of extracellular matrix-degrading metalloproteinases (MMPs). This review summarizes the evidence that implicates ROS as key regulators of MMP production and the importance of these interactions in disease pathologies.     

Permeability to cobalt ions and action of colicin K in a mutant that overproduces cardiolipin

A mutant of Escherichia coli that produces excess cardiolipin becomes less capable of transporting Co²⁺. Cardiolipin therefore does not act as an ionophore under these conditions. Colicin K brings about the typical increase in permeability to Co²⁺ in the mutant.     

Mitochondrial redox biology and homeostasis in plants

Mitochondria are key players in plant cell redox homeostasis and signalling. Earlier concepts that regarded mitochondria as secondary to chloroplasts as the powerhouses of photosynthetic cells, with roles in cell proliferation, death and ageing described largely by analogy to animal paradigms, have been replaced by the new philosophy of integrated cellular energy and redox metabolism involving mitochondria and chloroplasts. Thanks to oxygenic photosynthesis, plant mitochondria often operate in an oxygen- and carbohydrate-rich environment. This rather unique environment necessitates extensive flexibility in electron transport pathways and associated NAD(P)-linked enzymes. In this review, mitochondrial redox metabolism is discussed in relation to the integrated cellular energy and redox function that controls plant cell biology and fate.