Magnesium magnetic isotope effect: A key to the mechanochemistry of phosphorylating enzymes as molecular machines

The discovery of the powerful magnesium isotope effect on enzymatic ATP synthesis provides a new insight into the mechanochemistry of enzymes as molecular machines. The catalytic activities of ATPase, creatine kinase, and glycerophsphate kinase containing a Mg²⁺ ion with magnetic isotope nuclei (²⁵Mg) were found to be two to four times higher than those of the enzymes with spinless, nonmagnetic magnesium cation isotopes (²⁴Mg or ²⁶Mg). This demonstrates unambiguously that ATP synthesis is a spin-selective process involving Mg2+ as the electron-accepting reagent. ATP synthesis proceeds in an ion-radical pair consisting of an ADP oxyradical and Mg²⁺. In this process, the magnesium bivalent cation is the key agent that transforms the mechanics of a protein molecule into chemical processes. This ion is the crucial structural component of enzymes as mechanochemical molecular machines.     

ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase

A modified ‘cold chase’ technique was used to study tight [¹⁴C]ADP and [¹⁴C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF₀F₁). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF₀F₁ or CF₁ reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k _d of about 5 min⁻¹. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF₀F₁. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF₁ dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF₁ becomes bound to a thylakoid membrane.     

Resonance phenomenon of the ATP motor as an ultrasensitive biosensor

We designed a rotary biosensor as a damping effector, with the rotation of the F₀F₁-ATPase driven by Adenosine Triphosphate (ATP) synthesis being indicated by the fluorescence intensity and a damping effect force being induced by the binding of an RNA molecule to its probe on the rotary biosensor. We found that the damping effect could contribute to the resonance phenomenon and energy transfer process of our rotary biosensor in the liquid phase. This result indicates that the ability of the rotary motor to operate in the vibration harmonic mode depends on the environmental conditions and mechanism in that a few molecules of the rotary biosensor could induce all of the sensor molecules to fluoresce together. These findings contribute to the theory study of the ATPase motor and future development of biosensors for ultrasensitive detection.     

Role of alphaPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase

The role of αPhe-291 residue in phosphate binding by Escherichia coli F₁F₀-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F₁F₀ membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.     

The molecular mechanism of ATP synthesis by F1F0-ATP synthase

ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F₁F₀-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.     

H+ transport and coupling by the F0 sector of the ATP synthase: Insights into the molecular mechanism of function

The F₀ sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F₁ sector, couples proton transport to ATP synthesis. The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on theEscherichia coli F₀ sector is reviewed here. TheE. coli F₀ is composed of three types of subunits (a, b, andc) and current information on their folding and organization in F₀ is reviewed. The structure of purified subunitc in chloroform-methanol-H₂O resembles that in native F₀, and progress in determining the structure by NMR methods is reviewed. Genetic experiments suggest that the two helices of subunitc must interact as a functional unit around an essential carboxyl group as protons are transported. In addition, a unique class of suppressor mutations identify a transmembrane helix of subunita that is proposed to interact with the bihelical unit of subunitc during proton transport. The role of multiple units of subunitc in coupling proton translocation to ATP synthesis is considered. The special roles of Asp61 of subunitc and Arg210 of subunita in proton translocation are also discussed.     

Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F₀F₁-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741–744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca²⁺-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (_M) and the Ca²⁺-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca²⁺ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.     

Knockdown of DAPIT (diabetes-associated protein in insulin-sensitive tissue) results in loss of ATP synthase in mitochondria

It was found recently that a diabetes-associated protein in insulin-sensitive tissue (DAPIT) is associated with mitochondrial ATP synthase. Here, we report that the suppressed expression of DAPIT in DAPIT-knockdown HeLa cells causes loss of the population of ATP synthase in mitochondria. Consequently, DAPIT-knockdown cells show smaller mitochondrial ATP synthesis activity, slower growth in normal medium, and poorer viability in glucose-free medium than the control cells. The mRNA levels of α- and β-subunits of ATP synthase remain unchanged by DAPIT knockdown. These results indicate a critical role of DAPIT in maintaining the ATP synthase population in mitochondria and raise an intriguing possibility of active role of DAPIT in cellular energy metabolism.     

gammaepsilon Sub-complex of thermophilic ATP synthase has the ability to bind ATP

The isolated epsilon subunit of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the epsilon subunit even in the ATP synthase complex should have the ability to bind ATP. To check if the ATP binding to the epsilon subunit within the ATP synthase complex may occur, the gammaepsilon sub-complex of TF(1) was prepared and ATP binding was examined. The results clearly showed that the gammaepsilon sub-complex can bind ATP.     

Kinetic studies of ATP synthase: The case for the positional change mechanism

The mitochondrial ATP synthases shares many structural and kinetic properties with bacterial and chloroplast ATP synthases. These enzymes transduce the energy contained in the membrane's electrochemical proton gradients into the energy required for synthesis of high-energy phosphate bonds. The unusual three-fold symmetry of the hydrophilic domain, F₁, of all these synthases is striking. Each F₁ has three identical subunits and three identical subunits as well as three additional subunits present as single copies. The catalytic site for synthesis is undoubtedly contained in the subunit or an , interface, and thus each enzyme appears to contain three identical catalytic sites. This review summarizes recent isotopic and kinetic evidence in favour of the concept, originally proposed by Boyer and coworkers, that energy from the proton gradient is exerted not directly for the reaction at the catalytic site, but rather to release product from a single catalytic site. A modification of this binding change hypotheses is favored by recent data which suggest that the binding change is due to a positional change in all three subunits relative to the remaining subunits of F₁ and F₀ and that the vector of rotation is influenced by energy. The positional change, or rotation, appears to be the slow step in the process of catalysis and it is accelerated in all F₁F₀ ATPases studied by substrate binding and by the proton gradient. However, in the mammalian mitochondrial enzyme, other types of allosteric rate regulation not yet fully elucidated seem important as well.     

Improved purification for thermophilic F1F0 ATP synthase using n-dodecyl beta-D-maltoside

Here we report a fast, simple purification for thermophilic F1F0 ATP synthase (TF1F0) that utilizes a cocktail of stabilizing reagents and the detergent n-dodecyl beta-D-maltoside to yield enzyme with an ATPase activity of 41 micromol/min/mg, 2.5-fold higher than that previously reported. ATPase activity was 80% inhibited by the F0-reactive reagent dicyclohexylcarbodiimide, indicating that F1-F0 interactions were largely intact. To measure ATP-driven proton pumping activity, purified TF1F0 was incorporated into liposomes, and the ATP-induced change in internal pH was measured using the fluorescent probe pyranine. In the presence of valinomycin, a maximum ATP-driven deltapH of 0.8 units was obtained. To measure ATP synthesis activity, TF1F0 was incorporated into liposomes with the light-dependent proton pump bacteriorhodopsin. Proteoliposomes were illuminated to generate an electrochemical gradient, after which ADP and inorganic phosphate were added to initiate ATP synthesis. A steady state ATP synthesis activity of 490 nmol/min/mg was achieved after an initial approximately 30-min lag phase.     

The Catalytic Transition State in ATP Synthase

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP–fluoroaluminate, Mg-ADP–fluoroscandium, and corresponding Mg-IDP–fluorometals as transition-state analogs; (2) fluorescence signals of -Trp331 and -Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of -Arg376, acting as an arginine finger residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site / subunit-interface conformational movement, which is linked to -subunit rotation.     

Frontiers in ATP synthase research: Understanding the relationship between subunit movements and ATP Synthesis

How biological systems make ATP has intrigued many scientists for well over half the 20th century, and because of the importance and complexity of the problem it seems likely to continue to be a source of fascination to both senior and younger investigators well into the 21st century. Scientific battles fought to unravel the vast secrets by which ATP synthases work have been fierce, and great victories have been short-lived, tempered with the realization that more structures are needed, additional subunits remain to be conquered, and that during ATP synthesis, not one, but several subunits may undergo either significant conformational changes, repositioning, or perhaps even physical rotation similar to bacterial flagella^(1,2). In this introductory article, the author briefly summarizes our current knowledge about the complex substructure of ATP synthases, what we have learned from X-ray crystallography of the F₁ unit, and current evidence for subunit movements.     

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.     

Coupling Proton Movement to ATP Synthesis in the Chloroplast ATP Synthase

The chloroplast F₀F₁-ATP synthase-ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the γ subunit of the chloroplast F₁ enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthases of higher plants and its physiological significance lies in preventing nonproductive depletion of essential ATP pools in the dark. The three-dimensional structure of the chloroplast F₁ gamma subunit has not yet been solved. To examine the mechanism of dithiol regulation, a model of the chloroplast gamma subunit was obtained through segmental homology modeling based on the known structures of the mitochondrial and bacterial γ subunits, together with de novo construction of the unknown regulatory domain. The model has provided considerable insight into how the dithiol might modulate catalytic function. This has, in turn, suggested a mechanism by which rotation of subunits in F₀, the transmembrane proton channel portion of the enzyme, can be coupled, via the ε subunit, to rotation of the γ subunit of F₁ to achieve the 120° (or 90°+30°) stepping action that is characteristic of F₁ γ subunit rotation.     

F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.     

Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase

The sodium ion-translocating F(1)F(0) ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits. The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy. Here, we present an alpha-carbon model of the c-subunit ring. Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop. The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of approximately 17A (1.7 nm). The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix. Each inner helix is connected by a clearly visible loop to an outer C-terminal helix. The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus. Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface. Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I.tartaricus c-rings compared to those of other organisms.     

Overexpression of a mitochondrial ATP synthase small subunit gene (AtMtATP6) confers tolerance to several abiotic stresses in Saccharomyces cerevisiae and Arabidopsis thaliana

Mitochondrial F(1)F(0)-ATPase is a key enzyme in plant metabolism, providing cells with ATP that uses the transmembrane electrochemical proton gradient to drive synthesis of ATP. A 6 kDa protein (At3g46430) has been previously purified from Arabidopsis thaliana mitochondrial F(1)F(0)-ATPase. In this study, the gene (AtMtATP6; GenBank accession no. AK117680) encoding this protein was isolated from Arabidopsis and characterized. Northern blot analyses showed that the expression of AtMtATP6 gene in Arabidopsis suspension-cultured cells was induced by several abiotic stresses from salts, drought, and cold. Over-expression of AtMtATP6 gene in transgenic yeast and Arabidopsis plants increased the resistance to salts, drought, oxidative and cold stresses. Taken together, our data raise the possibility that induction of the F(1)F(0)-ATPase plays a role in stress tolerance.     

Organization and nucleotide sequence of the atp genes encoding the ATP synthase from alkaliphilic Bacillus firmus OF4

Summary The atp operon from the extreme alkaliphile Bacillus firmus OF4 was cloned and sequenced, and shown to contain genes for the eight structural subunits of the ATP synthase, preceded by a ninth gene predicted to encode a 14 kDa hydrophobic protein. The arrangement of genes is identical to that of the atp operons from Escherichia coli, Bacillus megaterium, and thermophilic Bacillus PS3. The deduced amino acid sequences of the subunits of the enzyme are also similar to their homologs in other ATP synthases, except for several unusual substitutions, particularly in the a and c subunits. These substitutions are in domains that have been implicated in the mechanism of proton translocation through F₀-ATPase, and therefore could contribute to the gating properties of the alkaliphile ATP synthase or its capacity for proton capture.     

Effect of cetyltrimethylammonium on ATP hydrolysis and proton translocation in the F0-F1 H+-ATP synthase of mitochondria

The amphiphylic alkyl cation cetyltrimethylammonium inhibits the catalytic activity of soluble and membrane-bound F₁ in a noncompetitive fashion. In sonic submitochondrial particles the Dixon plot showed a peculiar pattern with upward deviation at cetyltrimethylammonium concentration higher than 80µM. In membrane-bound F₁ the inhibition by cetyltrimethylammonium was potentiated by the F₀ inhibitor ologomycin. Cetyltrimethylammonium also inhibited the oligomycin-sensitive proton conductivity in F₁-containing particles but was without any effect in F₁-depleted particles. Also this inhibitory effect was potentiated by oligomycin. These results indicate functional cooperative interactions between F₀ and F₁.