Mechanism Of Oxidative Phosphorylation Research Articles

Correction: Studies on the Mechanism of Oxidative Phosphorylation: Effects of Specific F0 Modifiers on Ligand-Induced Conformation Changes of F1

Correction: Studies on the Mechanism of Oxidative Phosphorylation: Effects of Specific F0 Modifiers on Ligand-Induced Conformation Changes of F1

Studies of the mechanism of oxidative phosphorylation: Effects of specific F 0 modifiers on ligand-induced conformation changes of F 1

Studies of the mechanism of oxidative phosphorylation: Effects of specific F ₀ modifiers on ligand-induced conformation changes of F ₁

Studies on the mechanism of oxidative phosphorylation: effects of specific F0 modifiers on ligand-induced conformation changes of F1.

Aurovertin is a fluorescent antibiotic that binds to the catalytic beta subunits of the mitochondrial F1-ATPase and inhibits ATP synthesis and hydrolysis. ATP, ADP, and membrane energization in submitochondrial particles (SMP) alter the fluorescence of F1-bound aurovertin. These fluorescence changes are considered to be in response to the conformation changes of F1-ATPase. This paper shows that the ATP-induced fluorescence change of aurovertin bound to SMP or complex V (purified ATP synthase complex F0-F1) is inhibited when these preparations are pretreated with oligomycin or N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition is not seen with isolated F1-ATPase. These and other results have suggested that modifications of the DCCD-binding protein in the membrane sector (F0) of the ATP synthase complex are communicated to F1, thereby altering the binding characteristics of ATP to the beta subunits. By analogy, it is proposed that modifications (e.g., protonation/deprotonation) of the DCCD-binding protein effected by protonic energy alter the conformation of F1 and bring about the substrate/product binding changes that appear to be essential features of the mechanism and regulation of oxidative phosphorylation.

Studies on the mechanism of oxidative phosphorylation. Catalytic site cooperativity in ATP synthesis.

Oxidative phosphorylation catalyzed by bovine heart submitochondrial particles appears to exhibit negative cooperativity with respect to [ADP] and positive cooperativity in catalysis. Eadie-Hofstee plots (v/[S]versus v) of the kinetics of oxidative phosphorylation at the variable ADP concentration range of 1-1200 microM were curvilinear and could be analyzed for two apparent KmADP values differing by one order of magnitude, and two apparent Vmax values. The KmADP values with either NADH or succinate as the respiratory substrate were in the ranges of 10 and 100 microM, and the Vmax values in nmol of ATP formed X min-1 (mg of protein)-1 were, respectively, 500 and 1840 when NADH was the oxidizable substrate, and 550 and 100 when succinate was the energy source. Site-site cooperativity of the ATP synthase, which is a central feature of current theories for the mechanism of oxidative phosphorylation, has been well-documented for ATP hydrolysis by isolated F1-ATPase, but never before demonstrated for mitochondrial ATP synthesis.

The F 1F 0-ATPase of Escherichia coli. The substitution of alanine by threonine at position 25 in the c-subunit affects function but not assembly

A mutant strain of Escherichia coli carrying a mutation in the uncE gene which codes for the c-subunit of the F 1F 0-ATPase has been isolated and examined. The mutant allele, designated uncE513, results in alanine at position 25 of the c-subunit being replaced by threonine. The mutant F 1F 0-ATPase appears to be fully assembled and is partially functional with respect to oxidative phosphorylation. The ATPase activity of membranes from the mutant strain is resistant to the inhibitor dicyclohexylcarbodiimide, but this is due to the F 1-ATPase being lost from the membranes in the presence of the inhibitor. Mutant membranes from which the F 1-ATPase has been removed have a greatly reduced proton permeability compared with similarly treated normal membranes. The results are discussed in relation to a previously proposed mechanism of oxidative phosphorylation.

Effect of thyroidectomy on oxidative phosphorylation mechanisms in rat liver mitochondria

(1) In the presence of succinate, 2,4-dinitrophenol raised the oxygen consumption rate of state 3 liver mitochondria, whatever the thyroid state. The rise was clearly greater in thyroidectomized than in normal rats, but the uncoupled state 3 mitochondria of thyroidectomized rats nevertheless consumed oxygen more slowly than normal rat mitochondria. Thyroidectomy therefore weakened respiratory chain activity and phosphorylation reactions. (2) With β-hydroxybutyrate as substrate, oxygen consumption V of state 3 mitochondria was greatly diminished in thyroidectomized rats, but the K M remained unchanged. In the presence of succinate, state 3 respiration was not affected by thyroidectomy when substrate concentrations were low, but diminished at concentrations above 3 mM. (3) Respiratory chain activity was estimated by determining the succinate cytochrome c reductase activity. After thyroidectomy, catalytic efficiency ( V/ K M ) dropped by 60% in intact mitochondria at high concentrations of the substrate, but only by 30% when concentrations were low. In submitochondrial particles, thyroidectomy reduced V without changing K M . These results suggest that in thyroidectomized rats, succinate penetration is faster when substrate concentrations are low. (4) Mg 2+-stimulated ATPase activity dropped by 20% in thyroidectomized rats. In the presence of increasing concentrations of oligomycin, inhibition of ATPase activity was greater in normal than in thyroidectomized rat mitochondria. Thyroidectomy reduced by over 30% the ATPase activity actually involved in oxidative phosphorylation.

On the enzymic mechanism of oxidative phosphorylation.

Oxidative phosphorylation, like substrate-level phosphorylation, involves oxidative conversion of inorganic phosphate to a reactive species followed by interaction of this species with enzyme-bound ADP to form enzyme-bound ATP. The reactive species in a phosphoryl ester in substrate-level phosphorylation and phosphonium ion of orthophosphate in oxidative phosphorylation. The coupled synthesis is mediated by a combination of two classical enzymes in substrate-level phosphorylation and by a set of energy-coupled enzymes in oxidative phosphorylation. The full range of experimental evidence supporting this proposed enzymic mechanism of oxidative phosphorylation is presented as well as the rationalization of phenomena that hitherto have eluded explanation.

Portasomes as Coupling Factors in Active Ion Transport and Oxidative Phosphorylation

SYNOPSIS. We propose that particles, 7–15 nm in diameter, observed on the apical plasma membranes of cation transporting cells of insect midgut, salivary glands, and Malpighian tubules are modified F1-F coupling complexes such as those found on phosphorylating membranes of mitochondria, chloroplasts, and bacteria. We suggest the generic term, portasome, to describe all of these particles and point out that they are located on the side of the membrane which is electronegative and has the low cation concentration, i.e. , on the input side in each case. Biophysical evidence identifies the portasome bearing membrane as the ion transporting membrane in several insect epithelia, some of which exhibit ion modulated ATPase activity. The activity of a K+-modulated ATPase from Manduca sexta midgut is increased in portasome enriched plasma membrane fractions. We propose that portasomes orient the scalar hydrolysis of negatively charged MgATP2− to less negatively charged MgADP thereby eliminating the attraction of MgATP2− to K+ with the result that the K+ ions are ejected to the opposite side of the portasome bearing membrane. This mechanism explains the coupling of the scalar hydrolysis of ATP to the vectorial active transport of K+ which leads to the establishment of a K+ electrochemical gradient. The reverse process, but with an H+ ionophore replacing a K+ ionophore in the portasome, would provide a mechanism for coupling the vectorial flow of H+, driven by a proton electrochemical gradient, to scalar ATP synthesis and thereby provide a mechanism for oxidative phosphorylation. Electrogenic active potassium ion transport would appear to have evolved from oxidative phosphorylation.

Restoration of active transport of solutes and oxidative phosphorylation by naphthoquinones in irradiated membrane vesicles from Mycobacterium phlei.

Irradiation of the inverted membrane vesicles of Mycobacterium phlei with light at 360 nm inactivated the natural menaquinone [MK(9)(II-H)] and resulted in a loss of substrate oxidation, pH gradient, membrane potential, active transport of proline or calcium ions, and oxidative phosphorylation. Restoration of the protonmotive force and active transport occurred on addition of naphthoquinones such as vitamin K(1), menadione, or lapachol to the irradiated membrane vesicles. However, coupled phosphorylation was restored only by vitamin K(1). Menadione and lapachol did not act as uncoupling agents. The magnitude of the pH gradient and membrane potential in the quinone-restored system was a reflection of the rate of oxidation and was correlated with the rate of uptake of proline or Ca(2+). These results are consistent with the chemosmotic hypothesis proposed for the energy transducing mechanism for active transport and further demonstrate that the complete respiratory chain is not required to drive active transport. In contrast, the data suggest that in addition to the driving force (protonmotive force) necessary to establish oxidative phosphorylation, a specific spatial orientation of the respiratory components, such as the naphthaquinones, is essential for the utilization of the proton gradient or membrane potential or both. Bypass of electrons from the respiratory chain with menadione may explain the inability of this quinone to restore oxidative phosphorylation; however, lapachol restores oxidation by the same electron transport pathway as the natural menaquinone but fails to restore phosphorylation. Because all three quinones restore the protonmotive force, other factors that are discussed must be considered in understanding the mechanism of oxidative phosphorylation.

Is the relationship between the plasma concentration of inorganic phosphate and the rate of oxygen consumption of significance in regulating energy metabolism in mammals?

Regulation of the energy metabolism in mammals ultimately depends on regulatory processes at the cellular level, which include a regulation of the oxidative phosphorylation in mitochondria. Although knowledge about the mechanisms of oxidative phosphorylation in isolated mitochondria has been rapidly increasing, our understanding of the regulation of energy metabolism at the cellular level is still only at its beginning, and, as stated already in 1947 [12], our concepts of the regulation of energy metabolism in the animal kingdom, as well as in human pathology, have hardly left the descriptive state. A review of the literature on the regulation of cellular and mitochondrial oxygen uptake as related to the level of extracellular inorganic phosphate, however, leads to the suggestion that the concentration of plasma Pi may well be essential for the regulation of energy metabolism in the animal kingdom as well as in humans in different clinical situations.

224 - Energy-transducing redox systems and the mechanism of oxidative phosphorylation

Photosynthesis consists essentially in the conversion of light energy into redox energy, and respiration in the subsequent conversion of redox energy into phosphate-bond energy. Based on experimental facts, it is proposed that the primary event in the biological transduction of redox energy into chemical-bond energy is the formation of a rich-energy compound in the broadest sense of the word (chemical, electronic, structural). The energy-transducing redox systems are constituted by two pairs of alternating potentials (depending on the activation state of one of the forms of the pair) and correspond to one of the following types: those which energize their reduced form, lowering the potential of the pair, and those which energize their oxidized form, increasing the potential of the pair. Water is a source of reducing power only in photosynthesis but not in respiration.

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations. 2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300 000. An inhibitor protein is bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment. 3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one. 4. Under de-energised conditions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate 32P i. 32P i is incorporated into the β and γ positions of the bound nucleotides, but β-labelling probably does not occur on the coupling ATPase. 5. Uncouplers inhibit the exchange of the free nucleotides or 32P i into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange. 6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.

Studies on the kinetic mechanism of oxidative phosphorylation.

The kinetics of the synthesis of ATP from ADP and Pi by beef heart submitochondrial particles were examined. When Pi was the variable substrate positive cooperativity was observed, whereas if ADP was varied, linear double reciprocal plots were obtained. The analog of Pi, thiophosphate, was a noncompetitive inhibitor of ATP synthesis with respect to ADP, while the analog of ADP, AMP (CH2)P, was an uncompetitive Pi leads to ATP exchange inhibitor. The kinetics of the initial velocity isotopic exchanges of oxidative phosphorylation were also examined. When the Pi leads to ATP exchange was examined, it was found that if ADP concentration was held constant while ATP and Pi concentrations were varied at a constant ratio, linear double reciprocal plots were obtained. However, if Pi concentration was held constant and ADP and ATP concentrations were varied at constant ratio, apparent substrate inhibition was observed. The 2, 4-dinitrophenol-sensitive ADP leads to ATP exchange showed linear double reciprocal plots regardless of which components were varied. These results are interpreted to indicate that in the direction of ATP synthesis, the reaction is ordered, with Pi adding to the enzyme before ADP addition.

Safranine as a probe of the mitochondrial membrane potential

One of the most central postulates of the chemiosmotic hypothesis of oxidative phosphorylation is the existence of a large electrochemical proton gradient (protonmotive force) across the mitochondrial membrane [ 1,2] . This gradient, which is composed of a membrane potential and a pH differential, is postulated to be the obligatory link between respiration and phosphorylation in the process of oxidative phosphorylation. The membrane potential has been suggested to be the major component of the electrochemical proton gradient under most conditions, and measurements of this potential are therefore essential for the understanding of the mechanism of oxidative phosphorylation and mitochondrial ion transport. The mitochondrial membrane potential has mainly been estimated from the distribution of K’ or Rb’ across the mitochondrial membrane in the presence of the ionophore valinomycin [3-g]. This method has given fairly consistent results and membrane potentials in the range 130190 mV (positive polarity extramitochondrially) have generally been reported in coupled mitochondria supplemented with either substrate or ATP. The differences in the values reported by different workers may for the most part be attributed to the use of different values for the matrix volume. The use of a method of this kind, which requires partially artificial experimental conditions, makes confirmation by other independent methods most desirable. This has prompted several workers to search for suitable spectroscopic membrane potential probes. The fluorescent probe MC V has been used by Chance and collaborators [9,10] as a membrane potential indicator in mitochondria, and Laris et al. [ 111 have employed a cyanine dye

Binding of adenine nucleotides to mitochondrial membrane

It has been reported from different laboratories [l-4] that mitochondria are capable of binding ATP. However the exact magnitude of binding, and the relevance of this process to the mechanism of oxidative phosphorylation are still unknown, due to the extremely low amounts of bound nucleotides, and to some difficulties in carrying out the measurements. The key to clarification of this problem has been the discovery in this laboratory [4] that HC104 treated mitochondria retain only a small portion of the originally bound ATP. This paper describes a method for quenching mitochondrial reactions which, by substituting HgClz for HClQ as the denaturating agent, easily permits to detect the binding of both ATP and ADP. The bound species can then be recovered, identified, and measured following acid extraction of the quenched mitochondrial pellet. Furthermore, the relationships between the binding of adenine nucleotides and the mechanism of oxidative phosphorylation are discussed. The results clearly show that the bound nucleotides are directly involved in the terminal step of phosphorylation of ADP.

The effects of cytochalasin-B on the membranes of enzymatically active mitochondria

Cytochalasin-B, one of a series of structurally related metabolites obtained from the moldHelminthosporium dematioideum, has been shown to alter several partial reaction parameters associated with mitochondrial oxidative phosphorylation. State-3 (active) respiratory rates, respiratory control ratios and overall ATPase activity were all inhibited by cytochalasin-B at concentrations between 0.2 and 1.0 mM. However, the efficiency of coupled oxidative phosphorylation, evaluated by the ADP/O ratio, was not significantly affected. Therefore, the metabolite does not appear to act at enzymic loci directly affiliated with the coupling mechanism of oxidative phosphorylation. The mode of action of cytochalasin-B may be conceived as restricting diffusion by means of macromolecular conformational changes occurring within the mitochondrial membranes. Electron micrographs of paired, 10 min mitochondrial incubations, in the presence and absence of 1.0 mM cytochalasin-B, clearly display a structural alteration within these organelles, such that upon the binding of cytochalasin-B the matrix area decreases and the inner membrane cristae develop condensed, tubular distortions.

Effects of respiratory chain inhibitors on mitochondrial ATPase activity

The effects of six respiratory chain inhibitors (amytal, rotenone, piericidin A, antimycin A, 2- N-heptyl-4-hydroxyquinoline N-oxide, and cyanide) on mitochondrial ATPase activity have been investigated. Each of these compounds inhibited the ATPase activity of intact mitochondria induced by uncoupling agents, ionophores, or alterations in ionic composition; the effects were variable depending upon the type and concentration of uncoupling agents or inhibitors utilized. The ATPase activity of sonicated submitochondrial particles was also diminished by respiratory inhibitors, but the isolated ATPase enzyme was not affected. We conclude from these results that these respiratory inhibitors interfere with the energy coupling mechanism of oxidative phosphorylation. The experimental observations tend to support the “chemical” theory, and appear to be less consistent with the “chemiosmotic” hypothesis of oxidative phosphorylation.

Role of a specific endogenous fatty acid fraction in the coupling-uncoupling mechanism of oxidative phosphorylation of brown adipose tissue

Sucrose isolated brown adipose tissue mitochondria are uncoupled. They contain between 120–150 nEq of free fatty acids per mg of protein. The removal by means of carnitine plus ATP of a minor fraction of these bound FFA transfers the mitochondria from an uncoupled to a coupled state. If the very last traces of this FFA fraction are removed the mitochondria resume all the characteristics of coupled mitochondria e.g. respiratory control, ATP-P i exchange and a DNP-stimulated ATPase and lack of endogenous Mg-requiring ATPase. Some characteristics of the respiratory system are also altered. The changes in many of the biochemical parameters are paralleled by changes in the ultrastructure of the mitochondria. The nature of the ATPase in freshly isolated mitochondria is that of a DNP- and not of a detergent-induced ATPase. The mechanism by which the endogenous mitochondrial FFA fraction exerts its action on mitochondrial ultrastructure is different from that of DNP. The differences observed in many parameters, including ultrastructure, between native and carnitine plus ATP recoupled mitochondria, suggest that the FFA effect is of an allosteric nature. The physiologic role of the described mechanism is discussed.

Quantitative structure-activity studies of hydrazones, uncouplers of oxidative phosphorylation.

α-Acyl-α-cyanocarbonyl-phenylhydrazones are effective uncouplers of oxidative phosphorylation. The pl50-values of a series of 60 hydrazones with substituent variations in six positions of the molecule were determined in rat liver mitochondria. They ranged from 4.96 to 7.06. The biological data together with physico-chemical compound and substituent parameters were analysed by multiple regression techniques to establish the structure activity relationship. The integral parameters pKa and log P (partition coefficient) gave correlations of only moderate significance. Good agreement of found and calculated pl50-values was obtained by an equation with electronic (σ) and hydrophobic (π) substituent parameters in linear and quadratic terms. It is concluded that the contribution of a substituent to uncoupling activity depends on its position in the molecule. The activity is enhanced by hydrophobic shielding of the acidic NH-group. The relevancy of these results in relation to current theories on the mechanism of oxidative phosphorylation is discussed.

Energy-producing metabolism of Tritrichomonas foetus: I. Evidence for control of intensity and the contribution of aerobiosis to total energy production

The oxygen consumption of Tritrichomonas foetus, calculated from the first 20 minutes of measurement, equalled approximately 33 μl/mg protein/hr. Although the glycogen content of the cell was high, the O 2 consumption was increased 2.23 times by glucose. After 90 minutes of anaerobic endogenous metabolism, the oxygen debt was not repaid, and the levels of ATP and ADP 1 1 Abbreviations used: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; IMP, inosine monophosphate; NAD, oxidized forms of nicotinamideadenine dinucleotide; NADH, reduced forms of nicotinamideadenine dinucleotide; 2, 4-DNP, 2,4-dinitrophenol; P O , phosphorylation quotient; NADPH, reduced form of nicotinamideadenine dinucleotide phosphate; FP, flavoprotein; SD, succinic dehydrogenase; TYM, cysteine, trypticase, yeast extract, maltose, serum medium. remained unchanged. When glucose was added after anaerobiosis, glycogen was rapidly resynthesized, and respiration was 1.20 times higher as compared with respiration in the presence of exogenous glucose in a suspension where no resynthesis occurred. The ratio of adenine nucleotides was markedly altered at the same time. These findings, together with the fact that dinitrophenol stimulated the respiration in the presence of glucose, might serve as evidence for respiratory control and indicate the possibility of oxidative phosphorylation in T. foetus. The significance of aerobic metabolism was investigated by measuring the anaerobic and aerobic glycogen consumption, which after 90 minutes attained a ratio of 7:5.15, and by estimating the decrease of the ATP level 15 minutes after anaerobic and aerobic resynthesis of glycogen when it reached the ratio of 7:4.82. Both results indicate that T. foetus has an oxidative phosphorylation mechanism and that the ratio of aerobic and anaerobic phosphorylations is 7:5. Spectrophotometric measurements give evidence for the presence of a flavine terminal oxidase. On the basis of P O calculations it seems probable that only half the oxidations in T. foetus are connected with phosphorylations.