BBA - Bioenergetics (v.1458, #2-3)

On what makes the γ subunit spin during ATP hydrolysis by F1 by Huimaio Ren; William S Allison (221-233).

A suggested minimal scheme for substrate binding by and interconversion of three forms of the catalytic sites of the ATP synthase is presented. Each binding change, that drives simultaneous interchange of the three catalytic site forms, requires a 120° rotation of the γ with respect to the β subunits. The binding of substrate(s) at two catalytic sites is regarded as sufficing for near maximal catalytic rates to be attained. Although three sites do not need to be filled for rapid catalysis, during rapid bisite catalysis some enzyme may be transiently present with three sites filled. Forms with preferential binding for ADP and Pi or for ATP are considered to arise from the transition state and participate in other steps of the catalysis. Intermediate forms and steps that may be involved are evaluated. Experimental evidence for energy-dependent steps and for control of coupling to proton translocation and transition state forms are reviewed. Impact of relevant past data on present understanding of catalytic events is considered. In synthesis a key step is suggested in which proton translocation begins to deform an open site so as to increase the affinity for ADP and Pi, that then bind and pass through the transition state, and yield tightly bound ATP in one binding change. ADP binding appears to be a key parameter controlling rotation during synthesis. In hydrolysis ATP binding to a loose site likely precedes any proton translocation, with proton movement occurring as the tight site form develops. Aspects needing further study are noted. Characteristics of the related MgADP inhibition of the F1 ATPases that have undermined many observations are summarized, and relations of three-site filling to catalysis are assessed.
Keywords: ATP synthase; Binding change; Rotational catalysis; Phosphorylation;

The ϵ subunit of bacterial and chloroplast F1F0 ATPases by Roderick A Capaldi; Birte Schulenberg (263-269).
Recent studies show that the ϵ subunit of bacterial and chloroplast F1F0 ATPases is a component of the central stalk that links the F1 and F0 parts. This subunit interacts with α, β and γ subunits of F1 and the c subunit ring of F0. Along with the γ subunit, ϵ is a part of the rotor that couples events at the three catalytic sites sequentially with proton translocation through the F0 part. Structural data on the ϵ subunit when separated from the complex and in situ are reviewed, and the functioning of this polypeptide in coupling within the ATP synthase is considered.
Keywords: F1F0 ATPase; ϵ Subunit; Rotary motor; Cross linking;

The F0F1 ATP synthase functions as a rotary motor where subunit rotation driven by a current of protons flowing through F0 drives the binding changes in F1 that are required for net ATP synthesis. Recent work that has led to the identification of components of the rotor and stator is reviewed. In addition, a model is proposed to describe the transmission of energy from four proton transport steps to the synthesis of one ATP. Finally, some of the requirements for efficient energy coupling by a rotary binding change mechanism are considered.
Keywords: F0F1 ATP synthase; Binding change mechanism; Rotational catalysis; Oxidative phosphorylation;

Synthase (H+ ATPase): coupling between catalysis, mechanical work, and proton translocation by Masamitsu Futai; Hiroshi Omote; Yoshihiro Sambongi; Yoh Wada (276-288).
Coupling with electrochemical proton gradient, ATP synthase (F0F1) synthesizes ATP from ADP and phosphate. Mutational studies on high-resolution structure have been useful in understanding this complicated membrane enzyme. We discuss mainly the mechanism of catalysis in the β subunit of F1 sector and roles of the γ subunit in energy coupling. The γ-subunit rotation during catalysis is also discussed.
Keywords: F0F1; Catalytic site; ATP synthesis; Proton transport; H+ ATPase; Rotational catalysis;

Molecular mechanisms of rotational catalysis in the F0F1 ATP synthase by Robert K Nakamoto; Christian J Ketchum; Phillip H Kuo; Yelena B Peskova; Marwan K Al-Shawi (289-299).
Rotation of the F0F1 ATP synthase γ subunit drives each of the three catalytic sites through their reaction pathways. The enzyme completes three cycles and synthesizes or hydrolyzes three ATP for each 360° rotation of the γ subunit. Mutagenesis studies have yielded considerable information on the roles of interactions between the rotor γ subunit and the catalytic β subunits. Amino acid substitutions, such as replacement of the conserved γMet-23 by Lys, cause altered interactions between γ and β subunits that have dramatic effects on the transition state of the steady state ATP synthesis and hydrolysis reactions. The mutations also perturb transmission of specific conformational information between subunits which is important for efficient conversion of energy between rotation and catalysis, and render the coupling between catalysis and transport inefficient. Amino acid replacements in the transport domain also affect the steady state catalytic transition state indicating that rotation is involved in coupling to transport.
Keywords: Adenosine triphosphate synthase; Coupling; F0F1; Mutational analysis; Subunit interaction; Transition state thermodynamics;

In ATP synthase, X-ray structures, demonstration of ATP-driven γ-subunit rotation, and tryptophan fluorescence techniques to determine catalytic site occupancy and nucleotide binding affinities have resulted in pronounced progress in understanding ATP hydrolysis, for which a mechanism is presented here. In contrast, ATP synthesis remains enigmatic. The molecular mechanism by which ADP is bound in presence of a high ATP/ADP concentration ratio is a fundamental unknown; similarly Pi binding is not understood. Techniques to measure catalytic site occupancy and ligand binding affinity changes during net ATP synthesis are much needed. Relation of these parameters to γ-rotation is a further goal. A speculative model for ATP synthesis is offered.
Keywords: ATP synthase; Proton pumping; Oxidative phosphorylation;

The Mg2+ cofactor of the F1F0 ATP synthase is required for the asymmetry of the catalytic sites that leads to the differences in affinity for nucleotides. Vanadyl (VIVO)2+ is a functional surrogate for Mg2+ in the F1-ATPase. The 51V-hyperfine parameters derived from EPR spectra of VO2+ bound to specific sites on the enzyme provide a direct probe of the metal ligands at each site. Site-directed mutations of residues that serve as metal ligands were found to cause measurable changes in the 51V-hyperfine parameters of the bound VO2+, thereby providing a means by which metal ligands were identified in the functional enzyme in several conformations. At the low-affinity catalytic site comparable to βE in mitochondrial F1, activation of the chloroplast F1-ATPase activity induces a conformational change that inserts the P-loop threonine and catch-loop tyrosine hydroxyl groups into the metal coordination sphere thereby displacing an amino group and the Walker homology B aspartate. Kinetic evidence suggests that coordination of this tyrosine by the metal when the empty site binds substrate may provide an escapement mechanism that allows the γ subunit to rotate and the conformation of the catalytic sites to change, thereby allowing rotation only when the catalytic sites are filled. In the high-affinity conformation analogous to the βDP site of mitochondrial F1, the catch-loop tyrosine has been displaced by carboxyl groups from the Walker homology B aspartate and from βE197 in Chlamydomonas CF1. Coordination of the metal by these carboxyl groups contributes significantly to the ability of the enzyme to bind the nucleotide with high affinity.
Keywords: F1F0 ATP synthase; F1 ATPase; Vanadyl;

Important subunit interactions in the chloroplast ATP synthase by Mark L. Richter; Ray Hein; Bernhard Huchzermeyer (326-342).
General structural features of the chloroplast ATP synthase are summarized highlighting differences between the chloroplast enzyme and other ATP synthases. Much of the review is focused on the important interactions between the ϵ and γ subunits of the chloroplast coupling factor 1 (CF1) which are involved in regulating the ATP hydrolytic activity of the enzyme and also in transferring energy from the membrane segment, chloroplast coupling factor 0 (CF0), to the catalytic sites on CF1. A simple model is presented which summarizes properties of three known states of activation of the membrane-bound form of CF1. The three states can be explained in terms of three different bound conformational states of the ϵ subunit. One of the three states, the fully active state, is only found in the membrane-bound form of CF1. The lack of this state in the isolated form of CF1, together with the confirmed presence of permanent asymmetry among the α, β and γ subunits of isolated CF1, indicate that ATP hydrolysis by isolated CF1 may involve only two of the three potential catalytic sites on the enzyme. Thus isolated CF1 may be different from other F1 enzymes in that it only operates on ‘two cylinders’ whereby the γ subunit does not rotate through a full 360° during the catalytic cycle. On the membrane in the presence of a light-induced proton gradient the enzyme assumes a conformation which may involve all three catalytic sites and a full 360° rotation of γ during catalysis.
Keywords: Chloroplast; ATP synthase; Bi-site mechanism;

The IF1 inhibitor protein of the mitochondrial F1F0-ATPase by David W Green; Gary J Grover (343-355).
Recent studies on the IF1 inhibitor protein of the mitochondrial F1F0-ATPase from molecular biochemistry to possible pathophysiological roles are reviewed. The apparent mechanism of IF1 inhibition of F1F0-ATPase activity and the biophysical conditions that influence IF1 activity are summarized. The amino acid sequences of human, bovine, rat and murine IF1 are compared and domains and residues implicated in IF1 function examined. Defining the minimal inhibitory sequence of IF1 and the role of conserved histidines and conformational changes using peptides or recombinant IF1 is reviewed. Luft’s disease, a mitochondrial myopathy where IF1 is absent, is described with respect to IF1 relevance to mitochondrial bioenergetics and clinical observations. The possible pathophysiological role of IF1 in conserving ATP under conditions where cells experience oxygen deprivation (tumor growth, myocardial ischemia) is evaluated. Finally, studies attempting to correlate IF1 activity to ATP conservation in myocardial ischemic preconditioning are compared.
Keywords: F1F0-ATPase inhibitor protein; IF1 inhibitor protein; Mitochondrial F1F0-ATPase; Mitochondria; Adenosine triphosphate; Ischemia;

The second stalk of Escherichia coli ATP synthase by Stanley D Dunn; Derek T McLachlin; Matthew Revington (356-363).
Two stalks link the F1 and F0 sectors of ATP synthase. The central stalk contains the γ and ϵ subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic α3β3 complex. The two b subunits and the δ subunit associate to form b 2δ, a second, peripheral stalk extending from the membrane up the side of α3β3 and binding to the N-terminal regions of the α subunits, which are approx. 125 Å from the membrane. This second stalk is essential for binding F1 to F0 and is believed to function as a stator during rotational catalysis. In vitro, b 2δ is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with δ. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one α subunit or αβ pair. However, the weak interactions between the polypeptides in b 2δ pose a challenge for the proposed stator function.
Keywords: Adenosine triphosphate synthase; Stator; b subunit; δ subunit; Stalk;

The ATP synthase of Escherichia coli: structure and function of F0 subunits by Gabriele Deckers-Hebestreit; Jörg-Christian Greie; Wolf-Dieter Stalz; Karlheinz Altendorf (364-373).
In this review we discuss recent work from our laboratory concerning the structure and/or function of the F0 subunits of the proton-translocating ATP synthase of Escherichia coli. For the topology of subunit a a brief discussion gives (i) a detailed picture of the C-terminal two-thirds of the protein with four transmembrane helices and the C terminus exposed to the cytoplasm and (ii) an evaluation of the controversial results obtained for the localization of the N-terminal region of subunit a including its consequences on the number of transmembrane helices. The structure of membrane-bound subunit b has been determined by circular dichroism spectroscopy to be at least 75% α-helical. For this purpose a method was developed, which allows the determination of the structure composition of membrane proteins in proteoliposomes. Subunit b was purified to homogeneity by preparative SDS gel electrophoresis, precipitated with acetone, and redissolved in cholate-containing buffer, thereby retaining its native conformation as shown by functional coreconstitution with an ac subcomplex. Monoclonal antibodies, which have their epitopes located within the hydrophilic loop region of subunit c, and the F1 part are bound simultaneously to the F0 complex without an effect on the function of F0, indicating that not all c subunits are involved in F1 interaction. Consequences on the coupling mechanism between ATP synthesis/hydrolysis and proton translocation are discussed.
Keywords: F0F1-ATPase; F0 complex; Topology; Monoclonal antibody; Circular dichroism; Escherichia coli;

Operation of the F0 motor of the ATP synthase by Peter Dimroth (374-386).
ATP, the universal carrier of cell energy is manufactured from ADP and phosphate by the enzyme ATP synthase using the energy stored in a transmembrane ion gradient. The two components of the ion gradient (ΔpH or ΔpNa+) and the electrical potential difference Δψ are thermodynamically but not kinetically equivalent. In contrast to accepted wisdom, the electrical component is kinetically indispensable not only for bacterial ATP synthases but also for that from chloroplasts. Recent biochemical studies with the Na+-translocating ATP synthase of Propionigenium modestum have given a good idea of the ion translocation pathway in the F0 motor. Taken together with biophysical data, the operating principles of the motor have been delineated.
Keywords: Adenosine triphosphate synthase; F0 motor; Ion translocation mechanism; Subunit c structure; Obligatory role of Δψ in ATP synthesis;

Structural interpretations of F0 rotary function in the Escherichia coli F1F0 ATP synthase by R.H Fillingame; W Jiang; O.Y Dmitriev; P.C Jones (387-403).
F1F0 ATP synthases are known to synthesize ATP by rotary catalysis in the F1 sector of the enzyme. Proton translocation through the F0 membrane sector is now proposed to drive rotation of an oligomer of c subunits, which in turn drives rotation of subunit γ in F1. The primary emphasis of this review will be on recent work from our laboratory on the structural organization of F0, which proves to be consistent with the concept of a c 12 oligomeric rotor. From the NMR structure of subunit c and cross-linking studies, we can now suggest a detailed model for the organization of the c 12 oligomer in F0 and some of the transmembrane interactions with subunits a and b. The structural model indicates that the H+-carrying carboxyl of subunit c is located between subunits of the c 12 oligomer and that two c subunits pack in a front-to-back manner to form the proton (cation) binding site. The proton carrying Asp61 side chain is occluded between subunits and access to it, for protonation and deprotonation via alternate entrance and exit half-channels, requires a swiveled opening of the packed c subunits and stepwise association with different transmembrane helices of subunit a. We suggest how some of the structural information can be incorporated into models of rotary movement of the c 12 oligomer during coupled synthesis of ATP in the F1 portion of the molecule.
Keywords: Adenosine triphosphate synthase; Proton transport; Rotary motor; Subunit c; F0 structure;

The electron microscopic data available on CF0F1 and its subcomplexes, CF0, CF1, subunit III complex are collected and the CF1 data are compared with the high resolution structure of MF1. The data are based on electron microscopic investigation of negatively stained isolated CF1, CF0F1 and subunit III complex. In addition, two-dimensional crystals of CF0F1 and CF0F1 reconstituted liposomes were investigated by cryo-electron microscopy. Progress in the interpretation of electron microscopic data from biological samples has been made with the introduction of image analysis. Multi-reference alignment and classification of images have led to the differentiation between different conformational states and to the detection of a second stalk. Recently, the calculation of three-dimensional maps from the class averages led to the understanding of the spatial organisation of the enzyme. Such three-dimensional maps give evidence of the existence of a third connection between the F0 part and F1 part.
Keywords: Chloroplast; H+-ATPase; Adenosine triphosphate synthase; H+-translocating ATPase from chloroplast; Electron microscopy;

Subunit c of the proton-transporting ATP synthase of Escherichia coli forms an oligomeric complex in the membrane domain that functions in transmembrane proton conduction. The arrangement of subunit c monomers in this oligomeric complex was studied by scanning mutagenesis. On the basis of these studies and structural information on subunit c, different molecular models for the potential arrangement of monomers in the c-oligomer are discussed. Intersubunit contacts in the F0 domain that have been analysed in the past by chemical modification and mutagenesis studies are summarised. Transient contacts of the c-oligomer with subunit a might play a crucial role in the mechanism of proton translocation. Schematic models presented by several authors that interpret proton transport in the F0 domain by a relative rotation of the c-subunit oligomer against subunit a are reviewed against the background of the molecular models of the oligomer.
Keywords: ATP synthase; F0 domain; Interacting subunit; Subunit c; Molecular model; Molecular mechanism; Proton translocation;

Development of an increasingly detailed understanding of the eucaryotic mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry, structure and function of F0 sector subunits in the contexts of the proton channel and the stator stalk. Still to be resolved are the precise locations and roles of other supernumerary subunits present in mitochondrial ATP synthase complexes, but not found in the bacterial or chloroplast enzymes. The highly developed system of molecular genetic manipulation available in the yeast Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene function based on the effects of gene disruption or deletion. In addition, the genes encoding ATP synthase subunits can be manipulated to introduce specific amino acids at desired positions within a subunit, or to add epitope or affinity tags at the C-terminus, enabling questions of stoichiometry, structure and function to be addressed. Newly emerging technologies, such as fusions of subunits with GFP are being applied to probe the dynamic interactions within mitochondrial ATP synthase, between ATP synthase complexes, and between ATP synthase and other mitochondrial enzyme complexes.
Keywords: Yeast mitochondrial ATP synthase; F0 subunit organization; Proton channel; Stator stalk; Stoichiometry; Green fluorescent protein fusion;

Organisation of the yeast ATP synthase F0:a study based on cysteine mutants, thiol modification and cross-linking reagents by Jean Velours; Patrick Paumard; Vincent Soubannier; Christelle Spannagel; Jacques Vaillier; Geneviève Arselin; Pierre-Vincent Graves (443-456).
A topological study of the yeast ATP synthase membranous domain was undertaken by means of chemical modifications and cross-linking experiments on the wild-type complex and on mutated enzymes obtained by site-directed mutagenesis of genes encoding ATP synthase subunits. The modification by non-permeant maleimide reagents of the Cys-54 of mutated subunit 4 (subunit b), of the Cys-23 in the N-terminus of subunit 6 (subunit a) and of the Cys-91 in the C-terminus of mutated subunit f demonstrated their location in the mitochondrial intermembrane space. Near-neighbour relationships between subunits of the complex were demonstrated by means of homobifunctional and heterobifunctional reagents. Our data suggest interactions between the first transmembranous α-helix of subunit 6, the two hydrophobic segments of subunit 4 and the unique membrane-spanning segments of subunits i and f. The amino acid residue 174 of subunit 4 is close to both oscp and the β-subunit, and the residue 209 is close to oscp. The dimerisation of subunit 4 in the membrane revealed that this component is located in the periphery of the enzyme and interacts with other ATP synthase complexes.
Keywords: Yeast; Mitochondria; ATP synthase; Subunit 4; Subunit 6; Subunit i; Subunit f; Subunit oscp; Cross-linking;

Most of what is known about the structure and function of subunit a, of the ATP synthase, has come from the construction and isolation of mutations, and their analysis in the context of the ATP synthase complex. Three classes of mutants will be considered in this review. (1) Cys substitutions have been used for structural analysis of subunit a, and its interactions with subunit c. (2) Functional residues have been identified by extensive mutagenesis. These studies have included the identification of second-site suppressors within subunit a. (3) Disruptive mutations include deletions at both termini, internal deletions, and single amino acid insertions. The results of these studies, in conjunction with information about subunits b and c, can be incorporated into a model for the mechanism of proton translocation in the Escherichia coli ATP synthase.
Keywords: ATP synthase; Proton translocation; Subunit a; Mutagenesis; F0 structure;

F0F1-ATP synthase: general structural features of ‘ATP-engine’ and a problem on free energy transduction by Eiro Muneyuki; Hiroyuki Noji; Toyoki Amano; Tomoko Masaike; Masasuke Yoshida (467-481).

ATP synthase comprises two rotary motors in one. The F1 motor can generate a mechanical torque using the hydrolysis energy of ATP. The Fo motor generates a rotary torque in the opposite direction, but it employs a transmembrane proton motive force. Each motor can be reversed: The Fo motor can drive the F1 motor in reverse to synthesize ATP, and the F1 motor can drive the Fo motor in reverse to pump protons. Thus ATP synthase exhibits two of the major energy transduction pathways employed by the cell to convert chemical energy into mechanical force. Here we show how a physical analysis of the F1 and Fo motors can provide a unified view of the mechanochemical principles underlying these energy transducers.
Keywords: ATP synthase; Bioenergetics; Mechanochemistry; Modeling; ATP Hydrolysis;