Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA.

Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed rho degrees cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in rho degrees cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4- for ADP3- by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1-ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian rho degrees cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in rho degrees cells and an incomplete FoF1-ATP synthase was also required. The maximum value of this potential was approximately 110 mV in permeabilized cells and approximately 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1-ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but approximately 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of rho degrees mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1-ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1-ATP synthase in maintaining the mitochondrial membrane potential in rho degrees cells.     

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.     

Inhibitory and Anchoring Domains in the ATPase Inhibitor Protein IF1 of Bovine Heart Mitochondrial ATP Synthase

The inhibitor protein IF1 is a basic protein of 84 residues which inhibits the ATPase activity of the mitochondrial FoF1-ATP synthase complex without having any effect on ATP synthesis. Results of cross-linking and limited proteolysis experiments are presented showing that in the intact FoF1 complex "in situ," in the inner membrane of bovine heart mitochondria, the central segment of IF1 (residues 42-58) binds to the alpha and beta subunits of F1 in a pH dependent process, and inhibits the ATPase activity. The C-terminal region of IF1 binds, simultaneously, to the OSCP subunit of Fo in a pH-independent process. This binding keeps IF1 anchored to the complex, both under inhibitory conditions, at acidic pH, and noninhibitory conditions at alkaline pH.     

Observations of rotation within the FoF1-ATP synthase: deciding between rotation of the Foc subunit ring and artifact

F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.     

Significance of the epsilon subunit in the thiol modulation of chloroplast ATP synthase

To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity.     

Nanoseconds molecular dynamics simulation of primary mechanical energy transfer steps in F1-ATP synthase

The mitochondrial membrane protein FoF1-ATP synthase synthesizes adenosine triphosphate (ATP), the universal currency of energy in the cell. This process involves mechanochemical energy transfer from a rotating asymmetric gamma-'stalk' to the three active sites of the F1 unit, which drives the bound ATP out of the binding pocket. Here, the primary structural changes associated with this energy transfer in F1-ATP synthase were studied with multi-nanosecond molecular dynamics simulations. By forced rotation of the gamma-stalk that mimics the effect of proton motive Fo-rotation during ATP synthesis, a time-resolved atomic model for the structural changes in the F1 part in terms of propagating conformational motions is obtained. For these, different time scales are found, which allows the separation of nanosecond from microsecond conformational motions. In the simulations, rotation of the gamma-stalk lowers the ATP affinity of the betaTP binding pocket and triggers fast, spontaneous closure of the empty betaE subunit. The simulations explain several mutation studies and the reduced hydrolysis rate of gamma-depleted F1-ATPase.     

Coupling of rotation and catalysis in F(1)-ATPase revealed by single-molecule imaging and manipulation

F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.     

A new solution structure of ATP synthase subunit c from thermophilic Bacillus PS3, suggesting a local conformational change for H+-translocation

In F(o)F(1)-ATP synthase, an oligomer ring of F(o)c subunits acts as a rotary proton channel of the F(o)-proton motor. On the basis of the solution structure of the Escherichia coli F(o)c (EF(o)c) monomer, the rotation of the C-terminal helix coupled with the reorientation of the essential Asp61 side-chain on deprotonation was proposed to drive rotation of the whole c-ring. We have determined the NMR structure of F(o)c from thermophilic Bacillus PS3, TF(o)c, in an organic solvent mixture (chloroform/methanol (3:1, v/v)). Our results showed that, independent of pH, the carboxyl group of the essential Glu56 of TF(o)c protrudes toward the outside of the hairpin, a third orientation that differs from either of the two orientations in EF(o)c. Therefore, it would be inappropriate to draw conclusions about the mechanism of c-ring rotation on the basis of the conformations observed only for EF(o)c. The appearance of different hairpin structures shows that there are multiple energy minima for the hairpin structure in terms of helix rotation and axial displacement. The multiple energy minima may also provide a base for the different oligomeric states in the c-ring structure. A rotation mechanism of the F(o) motor coupled with H(+)-translocation is discussed on the basis of these results and the recently reported crystal structure of the c-ring from Ilyobacter tartaricus Na(+)-ATPase.     

F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits

F1-ATPase, the catalytic part of FoF1-ATP synthase, rotates the central gamma subunit within the alpha3beta3 cylinder in 120 degrees steps, each step consuming a single ATP molecule. However, how the catalytic activity of each beta subunit is coordinated with the other two beta subunits to drive rotation remains unknown. Here we show that hybrid F1 containing one or two mutant beta subunits with altered catalytic kinetics rotates in an asymmetric stepwise fashion. Analysis of the rotations reveals that for any given beta subunit, the subunit binds ATP at 0 degrees, cleaves ATP at approximately 200 degrees and carries out a third catalytic event at approximately 320 degrees. This demonstrates the concerted nature of the F1 complex activity, where all three beta subunits participate to drive each 120 degrees rotation of the gamma subunit with a 120 degrees phase difference, a process we describe as a 'sequential three-site mechanism'.     

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.     

Evolutonary modifications of molecular structure of ATP-synthase gamma-subunit

The ATP-synthase gamma-subunit (FoF1) belongs to the rotor part of this oligomeric complex. Catalytic hydrolysis of adenosine triphosphate (ATP) is accompanied by rotation of gamma-polypeptide inside the sphere formed by six subunits (alphabeta)3 of the enzyme. The gamma-subunit regulates ATPase and ATP-synthase activities of the FoF1. In the present work, evolutionary and reverse changes of this regulatory polypeptide and their effect on properties of the enzyme are studied. It is suggested that elongation of the gamma-subunit globular part had resulted from the atpC intragene duplication in the process of adaptive evolution. The evolved fragment participates in light regulation of the chloroplast ATP-synthase.     

ATP Photosynthetic vesicles for light-driven bioprocesses

We prepared ATP photosynthetic vesicles from inside-out membranes of Escherichia coli cells that express delta-rhodopsin (a novel light-driven H⁺ transporter) and TF₀F₁-ATP synthase (a thermo-stable ATP synthase). These vesicles showed light-dependent ATP synthesis. Furthermore, coupling the ATP photosynthetic vesicles with an ATP-hydrolyzing hexokinase enabled light-dependent glucose consumption. The ATP photosynthetic vesicles indicate their potential to applied to light-driven ATP-regenerating bioprocess for various ATP-hydrolyzing bioproductions.     

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.     

Evolutionary modifications of molecular structure of ATP-synthase γ-subunit

The ATP-synthase γ-subunit (FoF1) belongs to the rotor part of this oligomeric complex. Catalytic hydrolysis of adenosine triphosphate (ATP) is accompanied by rotation of γ-polypeptide inside the sphere formed by six subunits (αβ)₃ of the enzyme. The γ-subunit regulates ATPase and ATP-synthase activities of the FoF1. In the present work, evolutionary and reverse changes of this regulatory polypeptide and their effect on properties of the enzyme are studied. It is suggested that elongation of the γ-subunit globular part had resulted from the atpC intragene duplication in the process of adaptive evolution. The evolved fragment participates in light regulation of the chloroplast ATP-synthase.     

An alternative reaction pathway of F1-ATPase suggested by rotation without 80 degrees/40 degrees substeps of a sluggish mutant at low ATP

F(1)-ATPase, a water-soluble portion of F(o)F(1)-ATP synthase, is a rotary motor driven by ATP hydrolysis. The central gamma-subunit rotates in the alpha(3)beta(3) cylinder by repeating four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, catalytic dwell, and rapid 40 degrees substep rotation. In the catalytic dwell, at least two catalytic reactions occur-cleavage of the enzyme-bound ATP and presumably release of the hydrolyzed product(s) from the enzyme-but we found that a slow ATP cleavage mutant of F(1)-ATPase from thermophilic Bacillus PS3 rotates at low ATP concentration without substeps and the catalytic dwell. Analysis indicates that in this alternative reaction pathway the two catalytic reactions occur during the preceding long ATP-binding dwell. Thus, F(1)-ATPase can operate through (at least) two competing reaction pathways, not necessarily through a simple consecutive reaction.     

ATP synthase that lacks F0a-subunit: isolation, properties, and indication of F0b2-subunits as an anchor rail of a rotating c-ring

In a rotary motor F1F0-ATP synthase, F0 works as a proton motor; the oligomer ring of F0c-subunits (c-ring) rotates relative to the F0ab2 domain as protons pass through F0 down the gradient. F0ab2 must exert dual functions during rotation, that is, sliding the c-ring (motor drive) while keeping the association with the c-ring (anchor rail). Here we have isolated thermophilic F1F0(-a) which lacks F0a. F1F0(-a) has no proton transport activity, and F0(-a) does not work as a proton channel. Interestingly, ATPase activity of F1F0(-a) is greatly suppressed, even though its F1 sector is intact. Most likely, F0b2 associates with the c-ring as an anchor rail in the intact F1F0; without F0a, this association prevents rotation of the c-ring (and hence the gamma-subunit), which disables ATP hydrolysis at F1. Functional F1F0 is easily reconstituted from purified F0a and F1F0(-a), and thus F0a can bind to its proper location on F1F0(-a) without a large rearrangement of other-subunits.     

Subunit rotation in F0F1-ATP synthases as a means of coupling proton transport through F0 to the binding changes in F1

The rotation of an asymmetric core of subunits in F₀F₁-ATP synthases has been proposed as a means of coupling the exergonic transport of protons through F₀ to the endergonic conformational changes in F₁ required for substrate binding and product release. Here we review earlier evidence both for and against subunit rotation and then discuss our most recent studies using reversible intersubunit disulfide cross-links to test for rotation. We conclude that the subunit of F₁ rotates relative to the surrounding catalytic subunits during catalytic turnover by both soluble F₁ and membrane-bound F₀F₁. Furthermore, the inhibition of this rotation by the modification of F₀ with DCCD suggests that rotation in F₁ is obligatorily coupled to rotation in F₀ as an integral part of the coupling mechanism.     

Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, F?rster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Movements of the epsilon-subunit during catalysis and activation in single membrane-bound H(+)-ATP synthase

F0F1-ATP synthases catalyze proton transport-coupled ATP synthesis in bacteria, chloroplasts, and mitochondria. In these complexes, the epsilon-subunit is involved in the catalytic reaction and the activation of the enzyme. Fluorescence-labeled F0F1 from Escherichia coli was incorporated into liposomes. Single-molecule fluorescence resonance energy transfer (FRET) revealed that the epsilon-subunit rotates stepwise showing three distinct distances to the b-subunits in the peripheral stalk. Rotation occurred in opposite directions during ATP synthesis and hydrolysis. Analysis of the dwell times of each FRET state revealed different reactivities of the three catalytic sites that depended on the relative orientation of epsilon during rotation. Proton transport through the enzyme in the absence of nucleotides led to conformational changes of epsilon. When the enzyme was inactive (i.e. in the absence of substrates or without membrane energization), three distances were found again, which differed from those of the active enzyme. The three states of the inactive enzyme were unequally populated. We conclude that the active-inactive transition was associated with a conformational change of epsilon within the central stalk.     

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F₁F_o-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F₁F_o-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F₁F_o-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F₁F_o-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F₁ catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F₁ to the lipid monolayer and the F_o membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F_o by electron microscopy and AFM.