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. This slippage decreases the efficiency of energy utilization but enables controlling and regulating metabolic demands.
Other non-coupled systems include the uncoupling proteins UCPs; Vercesi et al. It prevents excessive proton pumping and thus buffers proton concentration for providing the optimal performance of the ATP synthase. In chloroplasts, there are many alternative sinks for electrons, and several of them are non-coupled with proton gradient Ivanov et al. However, these pathways are important mainly in preventing overreduction of chloroplast ETC and their capacity is insufficient for fine-tuning of redox and energy balance in the whole cell.
We have presented evidence in this paper that the steady fluxes of adenylates, magnesium, hydrogen ions and phosphate established via thermodynamic buffering and regulated uncoupling support optimal load and consumption of ATP synthase and provide its stable catalytic cycle. AK equilibrium represents an essential bioenergetic regulatory principle for the maintenance of steady regimes of ATP synthesis in mitochondria and chloroplasts and its utilization in metabolic processes.
Despite of all similarities and differences in molecular regulation of ATP synthases in both mitochondria and chloroplasts, even though the topology is totally different, and despite the different location of AK in chloroplasts and mitochondria, in both cases the activities of ATP synthases are finely optimized. This optimization provides a dynamically stable homeostatic state essential for the maintenance of photosynthesis and for support of metabolic processes in plant cells and tissues.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Blair, J. Magnesium, potassium, and the adenylate kinase equilibrium. Magnesium as a feedback signal from the adenine nucleotide pool.
Blum, D. Biochemistry 51, — Blumenfeld, L. Physics of Bioenergetic Processes. Berlin: Springer. Google Scholar. Boyer, P. A perspective of the binding change mechanism for ATP synthesis. The mitochondrial genome retains similarity to its prokaryotic ancestor, as does some of the machinery mitochondria use to synthesize proteins.
In addition, some of the codons that mitochondria use to specify amino acids differ from the standard eukaryotic codons. Still, the vast majority of mitochondrial proteins are synthesized from nuclear genes and transported into the mitochondria. These include the enzymes required for the citric acid cycle, the proteins involved in DNA replication and transcription, and ribosomal proteins. The protein complexes of the respiratory chain are a mixture of proteins encoded by mitochondrial genes and proteins encoded by nuclear genes.
Proteins in both the outer and inner mitochondrial membranes help transport newly synthesized, unfolded proteins from the cytoplasm into the matrix, where folding ensues Figure 3. 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.
This causes diffusion of the tethered protein and its receptor through the membrane to a contact site, where translocator proteins line up green. When at this contact site, the receptor protein hands off the tethered protein to the translocator protein, which then channels the unfolded protein past both the inner and outer mitochondrial membranes. Figure Detail. Mitochondria cannot be made "from scratch" because they need both mitochondrial and nuclear gene products.
These organelles replicate by dividing in two, using a process similar to the simple, asexual form of cell division employed by bacteria. Video microscopy shows that mitochondria are incredibly dynamic. They are constantly dividing, fusing, and changing shape. Indeed, a single mitochondrion may contain multiple copies of its genome at any given time.
This page appears in the following eBook. Aa Aa Aa. 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? By comparing the positions of equivalent c -subunits in different rotational states, the observed rotational step sizes in the three rotational states of the ATP synthase appear to be almost exactly 3, 4 and 3 c -subunits Figure 2B. At the present resolution, the structures of subunit a and the c- ring do not appear to differ between rotary states. Similar integer step sizes were found in yeast ATP synthase Vinothkumar et al.
However, non-integer steps were seen in the chloroplast 14 c -subunits Hahn et al. B Top view of the c -ring and subunit a of the three rotational states from the cytoplasm when the F 1 regions of the three states are aligned. The b subunits appear to be the most flexible part of the enzyme. This video cannot be played in place because your browser does support HTML5 video.
You may still download the video for offline viewing. Alternatively, flexibility in the enzyme could maintain a constant rotational velocity. In Bacillus PS3 ATP synthase, the peripheral stalk is structurally simpler and more flexible than in yeast mitochondria Srivastava et al.
Given that these structures represent resting states of the bacterial ATP synthase, additional subunits, such as those in the central stalk, may show flexibility while under strain during rotation. Subunit a and the first copy of subunit b occupy the same positions as their yeast counterparts, while the second copy of subunit b is found at a position equivalent to subunit 8 in the yeast enzyme, which is known as A6L in mammals.
Atomic models for ATP synthase from mitochondria Guo et al. Subunit a from Bacillus PS3 shares The sequence for this loop varies significantly among species, suggesting that it is unlikely to be involved in the core function of proton translocation, despite being proximal to the periplasmic proton half-channel.
The loop forms an additional interface with subunit b near the periplasmic side of the membrane region and may interact with the N terminus of subunit b in the periplasm as well.
The structure suggests that two interfaces are necessary for subunits a and b to maintain a stable interaction. B Cross sections through a surface representation of the F O region simulated with rolling of a 1. The proton then rotates with the c -ring until it reaches the cytoplasmic half-channel formed between subunit a and the c -ring.
In the cytoplasmic half-channel, the proton is released from the Glu residue due to its interaction with the positively charged Arg of subunit a.
A Glu 56 residue from each protomer of the c -ring is shown. Arg is in purple, important residues for proton translocation identified by mutagenesis in E. The Bacillus PS3 ATP synthase structure implies a path for proton translocation through the bacterial complex involving two half-channels similar to the paths described for the mitochondrial and chloroplast enzymes. The cytoplasmic half-channel consists of an aqueous cavity at the interface of subunit a and the c -ring Figure 4B , left.
In the atomic model, both channels are visible when modeling the surface with a 1. The channels are wide and hydrophilic, suggesting that water molecules could pass freely through each of the channels before accessing the conserved Glu 56 of the c -subunits. During ATP synthesis, protons travel to the middle of the c -ring via the periplasmic half-channel and bind to the Glu 56 residue of a subunit c Figure 4C.
Protonation of the glutamate allows rotation of the ring counter-clockwise, when viewed from F 1 toward F O , delivering the subunit c into the hydrophobic lipid bilayer.
Protonation of the remaining nine subunits in the c -ring returns the first glutamate to subunit a , now into the cytoplasmic half-channel, where it releases its proton to the cytoplasm due to interaction with the positively charged Arg of subunit a. The proposed channels are consistent with a series of experiments probing water accessibility of residues in the E.
Together, these results suggest that ion selectivity in ATP synthases is probably determined by the c -ring, not subunit a.
In eukaryotes, subunit a is encoded by the mitochondrial genome, limiting genetic interrogation of the roles of different residues. In contrast, numerous mutagenesis studies have been performed on bacterial subunits a and b , with E.
A single G9D mutation in the E. Therefore, the G9D mutation in E. Cross-linking experiments suggested that the N terminus of the two copies of subunit b are in close proximity to each other Dmitriev et al. Recent structures of rotary ATPases suggest that the importance of this residue derives from its role in releasing protons bound to the Glu residues of the c -subunits as they enter the cytoplasmic half-channel, as well as preventing short-circuiting of the proton path by protons flowing between half-channels without rotation of the c -ring Morales-Rios et al.
Other residues in the E. Extensive mutations of E. Therefore, the negative surface charge from Glu Glu near the cytoplasmic half-channel facilitates proton transport across the lipid bilayer. The atomic model of subunit a also suggests that other residues such as Bacillus PS3 Thr , Asn , Glu , Tyr , and His , which are close to the cytoplasmic half-channel, may contribute to channel formation.
Many functional residues identified by mutagenesis are clustered around the periplasmic half-channel. In the atomic model of the Bacillus PS3 subunit a , Asp 19 and Glu are close to the periplasm, while Ser , Asn , and Gln are deeper inside the membrane. Among these residues, Glu and Ser are considered to be more important to enzyme function than Asn and Gln , as mutations of corresponding residues in E. These residues do not appear to be close enough to form a hydrogen bond in the S.
In Bacillus PS3 subunit a , the His residue is replaced by a serine Ser that similarly does not appear to close enough to Glu to form a hydrogen bond. Interestingly, although many of these functional residues appear important, their mutation to amino acids that cannot be protonated or deprotonated often does not completely abolish proton translocation Vik et al. The core itself is powered by the proton motive force conferred by protons crossing the mitochondrial membrane.
The binding-change mechanism as seen from the top of the F 1 complex. There are three catalytic sites in three different conformations: loose, open, and tight. As a result, ATP is released from the enzyme. In step 2, substrate again binds to the open site, and another ATP is synthesized at the tight site [ 25 ]. Masamitsu et. Conformational transitions that are significant in rotational catalysis are directed by the passage of protons through the F 0 assembly of ATP synthase.
On the other hand, when the proton concentration is higher in the mitochondrial matrix, the F 1 motor reverses the F 0 motor bringing about the hydrolysis of ATP to power translocation of protons to the other side of membrane. A team of Japanese scientists have succeeded in attaching magnetic beads to the stalks of F 1 -ATPase isolated in vitro , which rotated in presence of a rotating magnetic field. Additionally, ATP was hydrolyzed when the stalks were rotated in the counterclockwise direction or when they were not rotated at all [ 26 ].
Defects or mutations in this enzyme are known to cause many diseases in humans. The first defect in ATP synthase was reported by Houstek et. It was postulated that mutations in some factors explicitly involved in the assembly of ATP synthase could have caused the defect [ 27 ].
Kucharczyk et. A mutation in one or many of the subunits in ATPase synthase can cause these diseases [ 28 ]. These diseases also result decrement in intermediary metabolism and functioning of the kidneys in removing acid from the body due to increased production of free oxygen radicals.
Dysfunction of F 1 specific nuclear encoded assembly factors causes selective ATPase deficiency [ 31 ]. Similar inborn defects in the mitochondrial F-ATP synthase, termed ATP synthase deficiency, have been noted where newborns die within few months or a year.
Current research on ATP synthase as a potential molecular target for the treatment for some human diseases have produced positive consequences. Recently, ATPase has emerged as appealing molecular target for the development of new treatment options for several diseases. ATP synthase is regarded as one of the oldest and most conserved enzymes in the molecular world and it has a complex structure with the possibility of inhibition by a number of inhibitors.
In addition, structure elucidation has opened new horizons for development of novel ATP synthase-directed agents with plausible therapeutic effects. More than natural and synthetic inhibitors have been classified to date, with reports of their known or proposed inhibitory sites and modes of action [ 30 ]. We look to explore a few important inhibitors of ATP synthase in this paper. A drug, diarylquinoline also known as TMC developed against tuberculosis is known to block the synthesis of ATP by targeting subunit c of ATP synthase of tuberculosis bacteria.
Another such diarylquinone, Bedaquiline, is used for the treatment of multidrug resistant tuberculosis. Among other ATP synthase inhibitors, Bz is proapoptotic and 1,4-benzodiazepine binds the oligomycin sensitivity conferring protein OSCP component resulting in the generation of superoxide and subsequent apoptosis [ 32 , 33 , 34 ].
Melittin, a cationic, amphiphilic polypeptide is yet another ATP synthase inhibitor with documented inhibition of catalytic activities in mitochondrial and chloroplast ATP synthases [ 35 ].
IF1 and oligomycin are two other important classes of ATPase inhibitors. Oligomycin, an antibiotic, blocks protein channel F 0 subunit and this inhibition eventually inhibits the electron transport chain. This further prevents protons from passing back into mitochondria, eventually ceasing the operations of the proton pump, as the gradients become too high for them to operate.
Several polyphenolic phytochemicals, such as quercetin and resveratrol, have been known to affect the activity ATPase. At decreased concentrations, it inhibits both soluble and insoluble mitochondrial ATPase.
However, it does not impact oxidative phosphorylation occurring in other mitochondrial entities [ 39 , 40 , 41 ]. This scheme is based on the binding change mechanism of ATP hydrolysis [ 36 ]. IF1 is a naturally occurring 9. Several other plant products also serve as ATPase inhibitors. Polyphenols and flavones has been found effective in the inhibition of bovine and porcine heart F 0 F 1 -ATPase [ 41 , 42 ]. Efrapeptins are peptides which are produced by fungi of the genus Tolypocladium that have antifungal, insecticidal and mitochondrial ATPase inhibitory activities [ 43 ].
The mode of inhibition is competitive with ADP and phosphate [ 30 ]. Another inhibitor piceatannol, a stilbenoid, has been found to inhibit the F-type ATPase preferably by targeting the F 1 subunit [ 39 ]. Another inhibitor of ATPase is bicarbonate. Bicarbonate anion acts as activator of ATP hydrolysis and Lodeyro et. This inhibition of ATP synthase activity was competitive with respect to ADP at low fixed phosphate concentration, mixed at high phosphate concentration and non-competitive towards Pi at any fixed ADP concentration [ 44 ].
Other inhibitors of ATPase are tenoxin, lecucinostatin, fluro-aluminate, dicyclohexyl-carbodimide and azide. Leucinostatins bind to the F 0 part of ATP synthases and inhibit oxidative phosphorylation in mitochondria and photophosphorylation in chloroplasts [ 46 ]. Dicyclohexylcarbodiimide DCCD reacts with the carboxyl group of the conserved acidic amino acid residue of subunit c at higher pH levels.
So this compound can be considered as an inhibitor of both F O and F 1. However, inhibition of F O is highly specific, well-defined, and requires a much lower concentration of the inhibitor [ 48 ]. The list of inhibitors that directly and indirectly inhibit the activity of ATP synthase includes, magnesium, bismuth subcitrate and omeprazole, ethidium bromide, adenylyl imidodiphosphate, arsenate, angiostatin and enterostatin, ossamycin, dequalinium and methionine, almitrine, apoptolidin, aurovertin and citreoviridin, rhodamines, venturicidin, estrogens, catechins, kaempferol, genistein, biochanin A, daidzein and continues to grow [ 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ].
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