How do atp synthase work

There is a poor correlation between genotype and phenotype for the m. Additional determinants may be responsible and are a plausible explanation for this finding. One of these determinants has been proposed to be the higher degree of oligomycin vulnerability in the mutant than in wild type Kucharczyk et al.

In tightly coupled mitochondria the oxygen consumption rate and ATP synthesis depend on each other Rak et al. Therefore it is not very surprising to see a decreased respiratory activity in many ATP synthase defective mutants Rak et al. LeuPro Kucharczyk et al. LeuArg Rak et al. LeuArg Kucharczyk et al. So it appears that complex IV is an important target for the co-regulation of electron transfer and ATP synthesis in mitochondria Kucharczyk et al.

Mutant cybrids had less than half of the steady-state content of ATP and a substantial higher basal level of reactive oxygen species ROS Sikorska et al. LeuPro results in a phenotype varying from mild learning difficulties and foot deformities, ataxia, an acute neurological presentation with complete recovery, to Leigh syndrome Moslemi et al.

LeuPro has been described in a patient with Leigh syndrome Moslemi et al. The pathogenic mechanism of the m. A patient suffering from bilateral striatal necrosis and harboring a m. This phenotype is less severe than found in typical Leigh disease De Meirleir et al. This mutation has been reported in a patient with seizures and lactic acidemia Seneca et al.

Phenotypical variations between patients harboring the same mtDNA mutation have classically been attributed to mtDNA heteroplasmy. However, a discrepancy between the levels of mutant mtDNA and disease severity is sometimes observed D'Aurelio et al. The mtDNA background has been shown to play an important role in modulating the biochemical defects and clinical outcome D'Aurelio et al. MT-ATP6 mutations m. The mitochondrial haplogroup J has been shown to be associated with longevity and protection against certain diseases Verny et al.

Vice versa, respiratory chain defects upstream of ATP synthase contribute to ATP synthesis impairment, worsening the biochemical and clinical phenotype, e. The germ-line mutation m. The mtDNA variants m. Hence, mtDNA background can explain the biochemical and clinical variations observed between patients with the same mutation and a comparable mutation load. In general, phenotypic variability is proposed to be the result of interactions between the casual genes, genetic background or modifier genes mitochondrial or nuclear , and probably environmental factors Carelli et al.

This homoplasmic mutation has been found in a year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy Jonckheere et al. Trp55X Jonckheere et al. This resulted in an improper assembly and a decreased activity of the complex V holoenzyme Jonckheere et al. This homoplasmic mutation has been found in four unrelated patients who presented as infants with isolated hypertrophic cardiomyopathy and congestive heart failure, evolving to multisystem disease Ware et al. Electron microscopy of muscle tissue showed increased variation in size and shape of mitochondria with dense parallel cristae in one patient Ware et al.

For A6L, m. Trp55Arg is a pathogenic missense mutation, replacing a hydrophobic tryptophan into a basic arginine in a conserved region of the subunit. In subunit a, the nucleotide alteration results in the change of the initiation methionine to threonine, but the effect of this mutation on the function of this subunit is not clear Ware et al.

He died at the age of 10 years. Brain MRI showed involvement of the interpeduncular nucleus, the central tegmental tract, the white matter and the cerebellum. This leucodystrophy phenotype differs from the other mutations described in MT-ATP8 , where cardiac involvement predominates. Leucodystrophy has been described in two siblings with Leigh syndrome harboring the m.

It should be noted that complex V enzyme analysis was not performed in this patient Mkaouar-Rebai et al. Trp94Arg of the nuclear encoded complex V assembly gene ATP12 has been found in a patient who presented with a severe neonatal encephalopathy and dysmorphic features, evolving to basal ganglia atrophy within months and death at the age of 14 months De Meirleir et al. The mutation resulted in a severely decreased complex V amount and activity.

It was hypothesized that the change of a neutral polar amino acid tryptophan into a basic one arginine resulted in an ATP12 protein that was no longer able to mediate F 1 assembly De Meirleir et al. Later, it has been shown in a yeast model that the p. Trp94Arg mutation decreases the solubility of the protein, implying that the primary impact of the mutation was a change in physical rather than functional parameters Meulemans et al.

Mitochondrial morphology has been studied in fibroblasts, but no alterations compared to control cells could be observed Meulemans et al. The TMEM70 gene encodes a mitochondrial protein, transmembrane protein A common splice site mutation and an isolated frameshift mutation have been described in the TMEM70 gene particularly in a homogeneous ethnic group Romanies , with a clinical phenotype characterized by neonatal mitochondrial encephalocardiomyopathy, lactic acidosis and dysmorphic features Cizkova et al.

The description of four novel mutations in the TMEM70 gene has confirmed and expanded this classical clinical picture with early onset cataract, gastrointestinal dysfunction, congenital hypertonia and a fetal presentation of the syndrome Spiegel et al.

One patient with a milder clinical phenotype carrying the common splice site mutation and a missense variant has also been described Shchelochkov et al. This phenotype corroborates with two reported patients who are compound heterozygous for the common splice site mutation and a frame-shift mutation in exon 1 Honzik et al.

The exact mechanism behind this remains to be elucidated, although it has been suggested that TMEM70 is involved in complex V biogenesis Cizkova et al. Recently, a homozygous missense mutation c. The patient was a 22 year old woman presenting with neonatal onset, lactic acidosis, 3-methylglutaconic aciduria, and mild mental retardation.

She developed peripheral neuropathy Mayr et al. The mutation caused a decrease in the amount and activity of holocomplex V. Remarkably, subunit c was found to accumulate, in contrast to the other investigated complex V subunits, and in contrast to what was found in patients with a mutation in the TMEM70 Honzik et al.

Current available treatment options for patients with mitochondrial diseases are mainly supportive. Therefore, a lot of effort is put into the search for both pharmacological and genetic approaches to cure this devastating group of disorders for reviews, see DiMauro et al.

Focusing here on complex V deficiency, current research has mainly covered the therapy of mtDNA mutations. As mentioned above, complex V mutations can increase ROS production which is deleterious for the cell.

It has been shown that they significantly improved mitochondrial respiration and ATP synthesis in these cells Mattiazzi et al. In yeast studies, it has been suggested that forcing substrate-level phosphorylation to work overtime may counteract the energy crisis due to OXPHOS impairment Schwimmer et al.

For the application of this approach in OXPHOS-deficient human cells, exogenous substrates capable of stimulating the Krebs cycle flux while at the same time removing the excess of reduced nicotinamide adenine dinucleotide NADH have been chosen Sgarbi et al. It has been demonstrated that homoplasmic cybrids harboring the m. This genetic approach aims to force a shift in heteroplasmy, reducing the ratio of mutant to wild-type genomes also called gene-shifting DiMauro et al.

Various methods can be applied to achieve this goal. Here, a normal version of a mutant mtDNA-encoded protein is imported into the nucleus. To be transported to the mitochondrion, it has to be provided with a mitochondrial targeting signal, of which the genetic sequence can be borrowed from another mtDNA-encoded protein. The biochemical defect in cybrid cell lines harboring the m.

The correction here implies the transfection of mammalian cells with either mitochondrial or nuclear genes from other organisms encoding the protein of interest DiMauro et al. This has also been applied to human cybrids harboring the m. Also this approach could correct the biochemical defect in these cells Ojaimi et al. For example, the m. The gene for Sma I was fused to a mitochondrial targeting sequence and expressed in heteroplasmic mutant cybrid cells, which lost mutant mtDNA and recovered biochemically Tanaka et al.

It has been shown that culturing heteroplasmic m. It has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease Sato et al. Briefly, this means that the pronucleus of an oocyte, or zygote, of a woman carrying mutated mtDNA could be transferred to a donor enucleated oocyte, or zygote, carrying wild type mtDNA DiMauro et al. In that way, the offspring will carry all the nuclear — and physiognomonic - traits of the parents, but not the mutated mtDNA of the mother DiMauro et al.

Two successful approaches have recently been described. It has been demonstrated in mature non-human primate oocytes Macaca mulatta that the mitochondrial genome can be efficiently replaced by spindle-chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg Tachibana et al. Subsequently, it was possible to have normal fertilization and embryo development. The offspring was healthy Tachibana et al.

This is essentially the same procedure, except that the nuclear material, both the male and female pronucleus, is removed after fertilization Tachibana et al. It has been shown that transfer of pronuclei between abnormally fertilized human zygotes resulted in minimal carry-over of donor zygote mtDNA and is compatible with onward development of the blastocyst stage in vitro Craven et al.

There have been few randomized controlled trials for the treatment of mitochondrial disease Chinnery et al. To date, there is no clear evidence supporting the use of pharmacological agents, non-pharmacological treatments vitamins and food supplements , and physical training in patients with mitochondrial disorders Chinnery et al. Although very promising, all genetic techniques are still in an experimental phase and different technical, ethical and safety issues still have to be solved DiMauro et al.

Nevertheless, they do allow cautious optimism for the future. Since current therapeutic options for mitochondrial diseases are insufficient, the possibility of prenatal diagnosis for fetuses at risk is a valuable alternative.

If it concerns a known nuclear genetic defect, the mutation can directly be searched for in fetal tissue. Second, the heteroplasmy level differs between tissues and in one tissue through time Poulton and Marchington In this context, the m. Finally, it is suggested that the heteroplasmy level remains stable after 10 weeks of gestation Steffann et al.

Hitherto termination of pregnancy has been preferred in case of intermediate mutant loads Steffann et al. Remarkably, the intermediate mutant loads question the observation that the m.

Post-zygotic drift might explain this discrepancy Steffann et al. The interpretation of PGD results nevertheless demands a known correlation between mutation load and clinical phenotype. In addition, caution is warranted since some pathogenic mutations could exhibit different segregation behavior Dean et al. In case the genetic examination of an index case has revealed no mutations in both mtDNA and nDNA, prenatal diagnosis could still be possible.

In Nijmegen, complex V activity can be measured spectrophotometrically in native chorionic villi, cultured chorionic cells or cultured amniotic cells if there is a clear isolated complex V deficiency in fibroblasts and muscle tissue or other tissue of the index patient Niers et al.

As mentioned briefly, most of the structure of the bovine mitochondrial enzyme has been resolved. The structure of the membrane extrinsic part of bovine ATP synthase is complete Rees et al.

The structure of the c-ring has been resolved recently Watt et al. The structures of the membrane domain of subunit b, subunit a, and the accessory subunits e, f, g, and A6L remain to be determined Rees et al. Still, understanding the enzyme fully at a molecular level will require further efforts, both experimental and theoretical for a review, see Junge et al.

Next to structure and function of the monocomplex, also the role of di- and oligomerization of complex V, shaping the inner mitochondrial membrane, has been addressed in many studies both in yeast and in mammalian mitochondria Paumard et al. The role of IF 1 in this process has been shown to be important Campanella et al. Mitochondria are unusual organelles. They act as the power plants of the cell, are surrounded by two membranes, and have their own genome. They also divide independently of the cell in which they reside, meaning mitochondrial replication is not coupled to cell division.

Some of these features are holdovers from the ancient ancestors of mitochondria, which were likely free-living prokaryotes. What Is the Origin of Mitochondria? Figure 1: A mitochondrion. What Is the Purpose of a Mitochondrial Membranes? Figure 2: The electrochemical proton gradient and ATP synthase.

At the inner mitochondrial membrane, a high energy electron is passed along an electron transport chain. Is the Mitochondrial Genome Still Functional? Figure 3: Protein import into a mitochondrion. A signal sequence at the tip of a protein blue recognizes a receptor protein pink on the outer mitochondrial membrane and sticks to it. Logically, mitochondria multiply when a the energy needs of a cell increase.

Therefore, power-hungry cells have more mitochondria than cells with lower energy needs. For example, repeatedly stimulating a muscle cell will spur the production of more mitochondria in that cell, to keep up with energy demand.

Mitochondria, the so-called "powerhouses" of cells, are unusual organelles in that they are surrounded by a double membrane and retain their own small genome. They also divide independently of the cell cycle by simple fission. Mitochondrial division is stimulated by energy demand, so cells with an increased need for energy contain greater numbers of these organelles than cells with lower energy needs.

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Mitochondrial diseases and ATPase defects of nuclear origin. Identification and validation of the mitochondrial F1F0-ATPase as the molecular target of the immunomodulatory benzodiazepine Bz Benzodiazepine-induced superoxide signalsB cell apoptosis: mechanistic insight and potential therapeutic utility.

The Journal of clinical investigation. Bz superoxide signals B cell apoptosis via Mcl-1, Bak, and Bax. Biochemical pharmacology. M Search in Google Scholar. The Journal of Biological Chemistry. Nature Structural and Molecular Biology. It was shown Ko et al. The reaction can be presented by the following equation one proton is the substrate, whereas other protons have catalytic function :.

The difference in pH between matrix and IMS results in deprotonation of phosphate and of ADP in the matrix side, facilitating the Mg-dependent mechanism.

Magnesium participating in ATP synthase catalysis exhibits a profound catalytic effect as shown by Buchachenko et al. The activity with 25 Mg, which has magnetic isotopic nucleus, is two to three times higher than with 24 Mg or 26 Mg isotopes, having spinless non-magnetic isotopic nuclei. This suggests that the ATP synthesis is a spin-dependent ion-radical process. The magnesium bivalent cation transforms the protein molecule mechanics into a chemical reaction Buchachenko et al.

Although this mechanism was suggested for the mitochondrial ATP synthase, potentially it can be generalized for all ATP synthases including the chloroplast and even for other Mg-dependent enzymes. For ATP synthase, maintenance of storage energy conformational relaxation state is supported via stable dynamic environment buffering.

Proton can be also considered as a substrate and, in addition, it provides symport of Pi during translocation through the membrane Figure 1. The binding of substrate releases energy for conformational relaxation Blumenfeld, , thus this release should be optimized by the rate of substrate supply called load in the thermodynamic buffering concept of Stucki, a , b.

It was experimentally shown that the oxidative phosphorylation obeys linear and symmetric relations between flows and forces Lemasters and Billica, It can operate at optimal efficiency only if the conductance of the load, i. To satisfy this condition and maintain a stable far from equilibrium regime, the reversible ATP-utilizing reaction catalyzed by AK acts as a thermodynamic buffer Stucki, b.

The AK which is compartmentalized in the IMS of mitochondria acts as a filter by which the adenylate concentration is adjusted to a correct value before being handed over by adenylate translocator Roberts et al. It acts as a linear energy converter maintaining the linearity of oxidative phosphorylation within a physiological range Igamberdiev and Kleczkowski, It may seem that thermodynamic buffer enzymes dissipate huge amounts of energy, in case of AK by consuming significant portion of ATP molecules without using them for chemical synthesis; however, this is a cost for providing high efficiency of operation of ATP synthase by keeping it in prolonged state of conformational relaxation where energy can be efficiently directed to the chemical work ATP synthesis.

Due to this, linear non-equilibrium thermodynamics becomes applicable, and the metabolic flux of ATP generation becomes derivable from the concentrations of nucleoside phosphates and metal ions established under such equilibrium. Thus thermodynamic buffering represents the basic regulatory principle for the maintenance of a stable far from equilibrium regime with the minimal production of entropy.

Filling buffer reservoirs corresponds to the accumulation of free energy and the buffering of energy intermediates is its most efficient source Shnoll, The futile equilibration of product with substrate may be considered as a price for the maintenance of the energy-efficient process. In an effort to examine, under multiple metabolic conditions, contributions of mitochondrial proteins to cellular ATP levels, screening of an RNAi library targeting over nuclear-encoded genes corresponding to mitochondria-localized proteins revealed that AK was a key regulator of ATP levels Lanning et al.

According to Dahnke and Tsai , K cat of AK is s —1 which is one order of magnitude higher than that of ATP synthase and this is essential for efficient equilibration of substrate and product as in the case of other enzymes Igamberdiev and Roussel, ; Bykova et al. Concentrations of other ions e. Concentration of AMP established in this equilibrium is the main factor shifting cytosolic metabolism toward either catabolic or anabolic processes via regulation of AMP-activated protein kinase, which in plants is called SnRK1 sucrose-non-fermentingrelated protein kinase-1; Figure 1.

An important prerequisite of stable operation of ATP synthase is its coordination with function of two translocators, the adenylate translocator and the phosphate translocator. These proteins operate electrogenically, and the adenylate translocator exchanges free adenylates, while the phosphate translocator exchanges free phosphate in the symport with proton or in the antiport with OH —.

The electrical currents measured with the reconstituted adenylate translocator demonstrate electrogenic translocation of adenylates and charge shift of reorienting carrier sites Klingenberg, The mitochondrial phosphate transporter makes it possible for a very rapid transport of most of the Pi used in ATP synthesis Ferreira and Pedersen, Since the inner membrane of mitochondria possesses electrical potential difference depending on the rate of proton pumping by electron transport, the adenylate transporter and other charge-moving processes, this affects the transport of adenylates and their equilibration by AK Igamberdiev and Kleczkowski, , In the absence of a membrane potential, the equilibrium concentrations of total adenylates will correspond to equimolar concentrations of free adenylates inside and outside mitochondria.

The involvement of AK in respiration is likely supported by apyrase, an Mg-dependent enzyme, which is ubiquitously distributed in different tissues and exists in several subcellular compartments, including a cytosol and IMS-confined isozymes Flores-Herrera et al. Other sources of AMP include reactions leading to the formation of CoA-derivatives, activation of amino acids for protein synthesis, or nucleotide pyrophosphatase Igamberdiev and Kleczkowski, Thus the bioenergetic function of mitochondria is controlled from the outside cytosol , whereas chloroplast appears as a more autonomous system supporting its ATP-generating function via the ratio of adenylates in its stroma.

The dynamic environment of ATP synthase in chloroplasts is established in a different and in most aspects opposite way as compared to mitochondria. ATP synthase receives protons from the thylakoid lumen Figure 2 , which has smaller volume as compared to the mitochondrial IMS, and its pH dropping to the values below 5 Oja et al.

The size of granal thylakoids was determined for Arabidopsis as 4 nm stacking repeat distance to 5 nm diameter in darkness, increasing to 19 nm in width and to 9—10 nm in diameter in the light Kirchhoff et al.

Although two chloroplast adenylate transporters were identified Mohlmann et al. Thus, it is quite certain that the stromal pool of adenylates is the sole source for AK-equilibrium governed delivery of ADP for ATP synthase reaction in chloroplasts. Figure 2. Abbreviations are the same as in Figure 1. TM, thylakoid membrane; TPT, triose phosphate transporter. There is no AK in thylakoid lumen, and the entire chloroplastic AK activity is confined to chloroplast stroma.

Lange et al. Whereas silencing of the gene for one of the chloroplastic AK had no effect on plant phenotype, the second chloroplast AK was essential for proper growth and development. Although to-date the importance of the first chloroplast AK isoform is not clear, the crucial role of the second is evident in providing proper chloroplast functioning and integrity.

Several key metabolic processes are strongly affected by AK, e. Thus, the AK reaction prevents over-accumulation of ATP, resulting in the balance of anabolic Calvin cycle, starch synthesis, lipid biosynthesis, etc.

Both in rice and Arabidopsis , SnRK1 critically influences stress-inducible gene expression and the induction of stress tolerance, and its activity modulates plant developmental processes from early seedling development through late senescence Cho et al. Plants carrying out C 4 metabolism e. This duality underscores different functions of chloroplastic ATP synthase in those cells. Whereas bundle sheath chloroplasts carry out the Calvin cycle and accumulate starch in the light, the mesophyll chloroplasts do not have Rubisco, and starch accumulation there ceases in mature leaves Weise et al.

Thus, in the mesophyll, the ATP formed by ATP-synthase must be linked to entirely different processes than in bundle sheath cells, and this occurs prominently by coupling to AK reaction. In C 4 plants, the activity of AK from mesophyll cell chloroplasts is many-fold higher than in bundle sheath cells Kleczkowski and Randall, and it is coupled to regeneration of phosphoenolpyruvate PEP , the primary CO 2 acceptor in C 4 photosynthesis Hatch, A special function of AK and pyruvate, Pi-dikinase is evident also in C 3 plants under anoxic conditions, where the joint operation of these enzymes provides an efficient use of PPi in addition to ATP as energy currency, thus avoiding drastic depletion of energy when mitochondrial respiration is suppressed by the lack of oxygen Igamberdiev and Kleczkowski, a , b.

Figure 3 shows how the interactions between chloroplasts and mitochondria involving also cytosol are optimized by operation of ATP synthase in the two compartments and by AK present in the chloroplast stroma and mitochondrial IMS. In mitochondria, on the other hand, equilibration of adenylates takes place in the IMS, i. The depots of magnesium stored in vacuoles and mitochondria contribute via corresponding transporters Shaul et al.

Figure 3. When it is not necessary for mitochondrial ATP synthase to further support ATP synthesis the non-coupled pathways of respiration become operative. The shifts in balance between the reactions of load and consumption that are beyond the buffering capacity of AK and related mechanisms can be adjusted via irreversible exergonic reactions that are not coupled to ATP synthesis.

They correspond to a slippage occurring when an enzyme passes a proton without ATP synthesis, e.