Which of the following statements about mitochondrial chemiosmosis is not true

Chemiosmosis, Mitochondrial Channels, and the PT

The four basic postulates of chemiosmosis are (1) that the membrane-located adenosine triphosphatase (ATPase) reversibly couples the translocation of protons across the membrane to the flow of anhydro-bond equivalents between water and the couple adenosine triphosphate (ATP)/(adenosine diphosphatase (ADP) + Pi); (2) that the membrane-located respiratory chain catalyzes the flow of reducing equivalents, coupling reversibly the translocation of protons across the membrane to the flow of reducing equivalents during oxido-reduction; (3) that the mitochondrial inner membrane has specific carriers allowing anion-OH– and cation-H+ exchanges that regulate the pH and osmotic differential across the membrane, and permit the flux of essential metabolites without collapse of the membrane potential; and (4) that the systems of the first three postulates are located in a specialized coupling membrane which has a low permeability to protons and to anions and cations generally. The fourth postulate was widely (and erroneously) implied to mean that the inner membrane did not possess cation channels. Given this view, prevailing as late as the 1990s, it comes as no surprise that the PTP, at an estimated diameter of 3 nm, did not attract too much interest in bioenergetics. Things have changed dramatically over the last 15 years, following the demonstration that the inner mitochondrial membrane does possess cation channels that are expectedly tightly regulated, and that a PT can occur in cells, tissues, and organisms where it plays a role in cell death, and possibly in Ca2+ homeostasis.

4 Osmotic swelling experiments

Arguably the emphasis on osmosis in ‘chemiosmosis’ also occurs in part because large-scale ion and weak acid/base mobility to and from bulk phases can be observed macroscopically via osmotic swelling experiments. The facts are as follows:

Cells and organelles scatter light because their complex refractive indices differ from those of the medium in which they are suspended (Davey & Kell, 1996; Salzman, Singham, Johnston, & Bohren, 1990; Salzman, Wilder, & Jett, 1979; Shapiro, 2003).

If a mitochondrion, a bacterial protoplast, or a chloroplast thylakoid (or granal stack) is suspended in isomolar medium and then the exterior solution is diluted with water, or indeed made more concentrated with substances that cross the cell membrane, osmosis will cause water to flow from the more dilute to the more concentrated phase (although possibly counter-intuitive, osmosis occurs simply because water flows thermodynamically down its own concentration gradient).

The light scattering will decrease because the refractive index difference becomes lower.

In extremum, the closed membranes will shatter, and the scattering will become much lower still.

If such substances do not cross the membrane the cells will also change their scattering if the exterior solution becomes hyperosmolar.

If two permeant ions of opposite charge are added to the outside at hyperosmolar concentrations they will both penetrate, be diluted with water, lower light scattering, and eventually cause membrane rupture.

If only one ion is ‘permeant’ (this is a relative term) a membrane potential (known as a ‘diffusion potential,’ see next subsection) will be set up that can only be dissipated by adding a permeant ion of the opposite charge (or a counterion of the same charge).

The same osmotic behavior is true of weak acids and bases that are considered to pass through membranes mainly in their uncharged forms.

Some typical traces are shown in Fig. 5 (redrawn from Kell, Peck, Rodger, & Morris, 1981).

Thus the swelling induced by KSCN (where K+ is relatively impermeant, SCN− relatively permeant, also chaotropic) is speeded enormously by the addition of valinomycin, a K+ uniporter (see Fig. 5) (Thomas, Jayatilaka, & Corry, 2013).

Similarly, the swelling induced by the K+ salt of a weak acid is stimulated massively by nigericin, which is a K+/H+ antiporter (see Figs. 6 and 7, which also shows other ion movements widely discussed in chemiosmotic coupling).

These methods indicate the nature—charged or otherwise—of the permeating species, but not their mechanism (transporter-mediated vs through the bilayer).

Incidentally, as is now known, water does not cross real biomembranes via the bilayer but via specialized water channels (aquaporins) (Agre, 2004; Benga, 2003).

As noted, we are not at all disputing the behavior and effects of these ionophores when it comes to osmotic swelling experiments. We simply point to the fact that osmotic swelling measurements measure what they say, and do not directly relate to electron transport-coupled phosphorylation. To imply that they do would be an elementary error of logic.

Scope of Chapter

In this chapter we will consider first the theory of chemiosmosis which forms a testable basis to explain how energy generation is coupled to ATP synthesis and utilization. This will be followed by a discussion of ATPases and energy generation by fermentation. Bacterial aerobic and anerobic respiration will be discussed with an emphasis on the terminal cytochrome oxidases in aerobic respiration denitrification in anaerobic respiration and ATPases. The main systems by which regulatory proteins control aerobic and anaerobic respiration are covered through examples of one and two component regulators, followed by consideration of the switch from aerobic to anaerobic metabolism in E. coli and Paracoccus denitrificans. At the end of the chapter some examples/case studies of energy metabolism of bacteria are provided and related to the medical interest in these bacteria.

4.2.2.4 Ech and Coo

An alternative to Rnf for coupling the oxidation/reduction of ferredoxin with chemiosmosis are the membrane-bound energy-conserving hydrogenases Ech and Coo. These closely related hydrogenases belong to the subgroup of multisubunit membrane-bound energy-conserving [NiFe] hydrogenases, which have subunits related to subunits of complex I, but do not interact with quinones (Fox, He, Shelver, Roberts, & Ludden, 1996; Hedderich & Forzi, 2005; Vignais & Billoud, 2007). They catalyse the reduction of H+ with ferredoxin coupled to chemiosmotic energy conservation, or reduction of ferredoxin with H2 driven by reverse electron transport (Meuer, Kuettner, Zhang, Hedderich, & Metcalf, 2002). In SRP, these hydrogenases are mostly present in the Desulfovibrionaceae (Pereira et al., 2011; Rodrigues, Valente, Pereira, Oliveira, & Rodrigues-Pousada, 2003). In D. vulgaris, the Coo hydrogenase is not regulated by CO (Rajeev et al., 2012), as reported for the enzyme from Rhodospirillum rubrum (Fox et al., 1996), and shows considerable expression during growth with lactate/sulphate, in contrast to Ech (Keller & Wall, 2011). During growth with hydrogen/sulphate, the expression of the ech genes is upregulated, while that of the coo genes is downregulated (relative to lactate/sulphate conditions) (Pereira, He, Valente, et al., 2008). The Coo hydrogenase was shown to be essential for syntrophic growth of D. vulgaris with a methanogen in the presence of lactate, but not for growth with lactate/sulphate (Walker, Stolyar, et al., 2009). In D. gigas, which only contains the Ech and the HynAB periplasmic NiFe hydrogenases, a ΔechBC strain was not affected in growth with lactate, H2 or pyruvate with sulphate or by fermentation (Morais-Silva, Santos, Rodrigues, Pereira, & Rodrigues-Pousada, 2013).

3.1.1 Ca2 + sequestration by CVC

The CV of Paramecium releases substantial amounts of Ca2 + (Stock et al., 2002). This is based on chemiosmosis as it strictly depends on the ΔH+ generated by the organellar H+-ATPase. This is derived from the fact that concanamycin B, an efficient H+-ATPase inhibitor in Paramecium (Grønlien et al., 2002), retards by about 10-fold the reestablishment of [Ca2 +]i homeostasis after a significant Ca2 + load (Plattner et al., 2012).

No Ca2 +-ATPase of the type sarcoplasmic/endoplasmic reticulum Ca2 +-ATPase has been detected in the CVC of Paramecium (Hauser et al., 1998, 2000) and the same is true for the PMCA (plasma membrane Ca2 +-ATPase) (Elwess and Van Houten, 1997). This differs from Dictyostelium where PAT1, a PMCA-type Ca2 + pump, occurs also in the CVC (Moniakis et al., 1995). However, PAT1 possesses no conserved autoinhibitory calmodulin-binding domain (Pittman, 2011). Considering on the one hand that the potential calmodulin-binding domain in the carboxy-terminal part of orthodox PMCA molecules differs in Dictyostelium PAT1 (Moniakis et al., 1995) and on the other hand the wide variability of calmodulin-binding sites, in general (Fraga et al., 2010), it remains open whether PAT1 has to be classified as a genuine or as an atypical PMCA. CaM-binding studies could give the answer. See also Section 3.2.5 for the effect on anticalmodulin drugs on CV performance.

An alternative way of Ca2 + sequestration is reported from the CVC of T. cruzi, that is, a H+-pyrophosphatase (H+-PPase, Montalvetti et al., 2004). However, proteomics analysis of CVC-enriched fractions also revealed H+-ATPase SU B (Ulrich et al., 2011). In this parasite, the situation may be different insofar as its CVC is assumed to receive membrane components by fusion with acidocalcisomes whose H+-PPase activity is well established (Docampo et al., 2005; Moreno and Docampo, 2009).

In summary, the H+-ATPase can be considered the only primary active transporter in the CVC of Paramecium. For the additional PMCA-type pump in Dictyostelium, it has to be analyzed to what extent it might support the organelle-resident H+-ATPase. The same is true of the H+-PPase in Trypanosoma.

Mitochondria are membrane-enclosed organelles regarded as ‘cell power plants.’ Mitochondria generate most of the ATP in cells via ATPase rotation driven by the proton flow across the inner membrane by a process called chemiosmosis. This generates electrical potential energy in the form of a pH gradient across this membrane. The energy is released as electrons flow though the electron transport chain. The chain consists of five proteins (four cytochromes) that oxidize NADH generated by the citric acid cycle to NAD+ and includes a terminal cytochrome oxidase that catalyzes oxygen reduction by four electrons to form H2O, which accounts for 90% of cellular oxygen uptake. It has been hypothesized that more than a billion years ago mitochondria originated from ancient bacteria that were engulfed by phagocytozing eukaryotes to create a symbiotic relationship. The inner membrane and matrix of mitochondria have some similar proteins and genetic machinery (encoding 37 mitochondrial genes) to that found in bacteria which contribute to the synthesis of some mitochondrial proteins. Human mitochondria contain 615 proteins mostly coded for by nuclear genes. The intermembrane space contains various proapoptotic or antiapoptotic proteins that function in programmed cell death. Mitochondrial poisons can act by inhibiting electron transport proteins that increase reduction of oxygen to cytotoxic reactive oxygen species (ROS), or act as ionophores disrupting the proton gradient, or inhibit ATPase. Other mitochondrial toxins undergo redox cycling catalyzed by reduced electron transport cytochrome proteins to form radicals that reduce oxygen to ROS. However, while low ROS levels function in signaling and regulating a variety of cellular functions, high ROS levels contribute to apoptosis, necrosis, or the aging process leading to mitophagy (mitochondria-specialized autophagy). The review particularly focuses on mitochondrial function impairment that contributes to disease pathology and apoptosis or necrotic cell death. The review also focuses on cell death induced by endogenous toxins generated by genetic inborn errors of metabolism or acquired by human diseases or animal disease models. Also reviewed is the impairment of mitochondrial function that contributes to toxic side effects of various drugs or the toxic anticancer action induced by various drugs used in cancer chemotherapy. The review also focuses on the effects at the molecular level of respiratory poisons or drugs that impair mitochondrial complexes I–V.

Introduction

Cellular respiration, as an important method of ATP generation, represents one of the most basic metabolic processes of organisms. The physics and chemistry of cellular respiration help define such things as where organisms live, ecosystem-level interactions between species, and the morphology of multicellularity. We define cellular respiration as ATP-generating pathways in which electron transport systems (ETSs; Figure 1) are coupled to the generation of ATP through a process known as chemiosmosis (Figure 2). In contrast, ATP generation can also involve substrate-level phosphorylation (SLP), a process that uses energy within substrates, such as carbohydrates, to couple phosphates onto ADP (Figure 3). This latter process is exemplified by several steps in the glycolytic pathway (Figure 4), which may also be linked to cellular respiration by supplying reduced substrate to ETSs. Cellular respiration may be considered ecologically in terms of energy movement within microbe-based ecosystems; in terms of its evolution and relative benefits as compared to, for example, SLP; and in terms of the tradeoffs involved in the resulting challenges of gas exchange (O2 and CO2), especially in larger organisms. In the next four sections of this article, we employ the terms ‘cellular respiration’ and ‘respiration’ synonymously. The final section considers respiration exclusively as gas exchange in the whole organism.

2.1.3 Oxidative phosphorylation

The third step within the entire process of rebuilding ATP is oxidative phosphorylation, where reduced coenzymes such as NADH and FADH2 are oxidized with the help of oxygen; this is why animals and humans have to respire. Oxidative phosphorylation also takes place in the mitochondria of a cell. Hydrogen is split into protons and electrons, and electrons are passed to oxygen and other inorganic compounds. In a series of reactions, electrons are passed from one carrier to another within the process of oxidative phosphorylation in what is known as the electron-transfer chain (ETC). Water is split off during this process and the reoxidation of NADH, as of well as FADH2, or the transfer of electrons from one electron carrier to the next, releases energy, which is ultimately utilized for the formation of ATP from ADP and phosphate. The process of ATP synthesis using ‘free energy’ obtained when electrons are passed to several carriers (ETC) is known as chemiosmosis. The actual point of the synthesis of ATP takes place when electrons pass the inner mitochondrial membrane. Energy is released within this process, resulting in the synthesis of ATP. Oxidative phosphorylation could be summarized in the following way: Reduced coenzymes NADH + FADH2 + oxygen → ETC → oxidized coenzymes NAD+ + FAD + water + free energy → ADP → ATP.

Thirty-two molecules of ATP are obtained during oxidative phosphorylation per molecule of glucose. Together with the two molecules of ATP resulting from glycolysis, as well as the two molecules of GTP from the Krebs cycle (which can be readily transformed into ATP and can be counted as ATP), 36 molecules of ATP are obtained in total per molecule of glucose.

Some enzymes can only function in conjunction with coenzymes. Coenzymes are not protein based and can be nucleotides, ions or vitamins and, when these nucleotides, ions or vitamins are bound loosely to an enzyme, a coenzyme is obtained. One of the most important tasks of a coenzyme is to carry over, or to pass on, hydrogen and electrons and energy within biochemical processes. Substances, such as nucleotides, act as carrier materials for hydrogen and electrons. Nucleotides such as NAD+ and FAD, both coenzymes, are reduced by the uptake of hydrogen, resulting in NADH as well as FADH2.

As explained above, in pre-slaughter muscle tissue, as long as the animal is alive and therefore breathing, the filaments actin and myosin slide into each other and muscle contraction, as well as relaxation, takes place promoted by aerobically formed ATP (Fig. 2.3). Availability and utilization of ATP break down the actomyosin complex, obtained during contraction, into the separate fibres of actin and myosin once again. The pH value of lean muscle tissue at this stage, in the living animal, is between 6.8 and 7.2. Pork fat exhibits a pH of 6.3–6.6 at the point of slaughter and beef lard a pH value of around 6.8.

2.1.3 Oxidative Phosphorylation

The third step within the entire process of rebuilding ATP is oxidative phosphorylation, where reduced coenzymes such as NADH and FADH2 are oxidized by the help of oxygen: this is why animals and humans have to respire. Oxidative phosphorylation also takes place in the mitochondria of a cell. Hydrogen is split into protons and electrons, and electrons are passed on to oxygen and other inorganic compounds. In a series of reactions, electrons are passed on from one carrier to another within the process of oxidative phosphorylation in what is known as the electron transfer chain (ETC). Water is split off during this process, and the reoxidation of NADH, as well as FADH2, or the transfer of electrons from one electron carrier to the next one, releases energy, which is ultimately used for the formation of ATP out of ADP and phosphate (P). The process of ATP synthesis using “free energy” obtained through the passing-on of electrons to several carriers (ETC) is known as chemiosmosis. The actual point of the synthesis of ATP takes place when electrons pass the inner mitochondrial membrane. Energy is released within this process, resulting in the synthesis of ATP. Oxidative phosphorylation could be summarized in the following way:

Reduced coenzymes NADH/FADH2 + oxygen ⇒ ETC ⇒ oxidized coenzymes NAD+, FAD + water + free energy ⇒ ADP ⇒ ATP.

Thirty-two molecules of ATP are obtained during oxidative phosphorylation per molecule of glucose. Together with the two molecules of ATP resulting from glycolysis, as well as the two molecules of GTP from the Krebs cycle (which can be readily transformed into ATP and can be counted as ATP), 36 molecules of ATP are obtained in total per molecule of glucose.

Some enzymes can only function in conjunction with coenzymes. Coenzymes are not protein based and can be nucleotides, ions, or vitamins, and when these nucleotides, ions, or vitamins are bound loosely to an enzyme, a coenzyme is obtained. One of the most important tasks of a coenzyme is the carrying-over, or passing-on, of hydrogen, electrons and energy within biochemical processes. Substances, such as nucleotides, act as carrier-materials for hydrogen and electrons. Nucleotides such as NAD+ and FAD, both coenzymes, are reduced by the uptake of hydrogen resulting in NADH as well as FADH2.

As explained earlier, in preslaughter muscle tissue, as long as the animal is alive and therefore breathing, the filaments actin and myosin slide into each other, and muscle contraction, as well as relaxation, takes place promoted by aerobically formed ATP. Availability and utilization of ATP break down the actomyosin complex, obtained during contraction, into the separate fibers of actin and myosin once again. The pH value of lean muscle tissue at this stage, in the living animal, is between 6.8 and 7.2. Pork fat exhibits a pH of 6.3–6.6 at point of slaughter, and beef lard, a pH value of around 6.8 (Fig. 2.3).

Early Findings on the Mechanism of the Uptake Process

In the wake of the early findings, mitochondrial Ca2+ transport rapidly became very popular, and became recognized as an alternative to ADP phosphorylation in the harvesting of the energy made available by the respiratory chain. For a long time, the findings on the process were interpreted within the framework of the chemical theory of energy transduction; in hindsight, it may seem surprising that the process was not immediately related to the then emerging chemiosmotic theory of energy transduction, for which it evidently provided essential support. Chemiosmosis, however, only crept into the field laboriously, and did not take it over until well into the 1960s. Meanwhile, numerous studies had established the essential phenomenology of the process; thus, the stoichiometry of Ca2+ uptake to oxygen consumption was measured, finding that two Ca2+ ions were required to elicit the same extra consumption of oxygen elicited by one molecule of ADP, a finding that clearly confirmed the earlier conclusion by B Chance that the Ca2+/O stoichiometry in the transition from resting (state 4) to activated (state 3) respiration was 2–3 times the ADP/O stoichiometry. It also became clear that in the absence of phosphate the small amounts of Ca2+ that were taken up were maintained within mitochondria in a dynamic steady state, in which the leak of Ca2+ was balanced by its reuptake during the phase of resting respiration that followed the activated phase during which the pulse of Ca2+ had been taken up. It was an important finding, as it established the concept of a continuous cycling of Ca2+ across the mitochondrial membrane (the mitochondrial Ca2+ cycle), its long-term storage in the matrix only occurring when phosphate was also present and taken up. The finding that mitochondria could dynamically take up and release Ca2+ had obvious interest in the light of the emerging importance of Ca2+ in the regulation of cell activities, and placed mitochondria in a central position as potential regulators of cell function. At that time, however, an element in the scenario was still missing: while the fourth portion of the cycling process was defined, the back portion, that is, the route (mechanism) for the release of Ca2+ was not, and was only discovered some years later.

The limited uptake of Ca2+ in the absence of phosphate was eventually used to test the proposal of an electrophoretic mechanism for the uptake of Ca2+, which would have been consistent with the increasingly popular chemiosmotic principles. Diffusion potentials were artificially imposed across the mitochondrial membrane, showing that Ca2+ indeed traversed the membrane in response to an electrochemical gradient. Uncertainties of the various types complicated experiments aimed at establishing whether the mechanism of the uptake process was purely electrophoretic or partially charge compensated, but the presence of an electrophoretic component in the uptake process became generally accepted. As a result, the process of Ca2+ transport, which had been initially described as an active uptake followed by a passive release, became an energetically downhill uptake process, followed by an energetically uphill release leg. The uptake leg of the process was postulated to occur on an electrophoretic uniporter, its study being aided greatly by the discovery of specific inhibitors, the most popular being the histochemical stain Ruthenium Red (about 20 years later a derivative, RU360, was introduced, which specifically inhibited the uptake of Ca2+ with an IC50 of 0.2–2.0 nM).