Atp Synthase — a Marvellous Rotary Engine of the Cell

REVIEWS ATP SYNTHASE — A MARVELLOUS ROTARY ENGINE OF THE CELL Masasuke Yoshida, Eiro Muneyuki and Toru Hisabori ATP synthase can be thought of as a complex of two motors — the ATP-driven F1 motor and the proton-driven Fo motor — that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale. ELECTROCHEMICAL POTENTIAL “All enzymes are beautiful, but ATP synthase is one of tale is still being unravelled. In this review, we focus on GRADIENT the most beautiful as well as one of the most unusual the mechanism of rotary catalysis of ATP synthase — When two aqueous phases are and important” said Paul Boyer1. ATP synthase — also that is, how rotation and catalysis are coupled and regu- separated by a membrane, the called the F F -ATP synthase or F F -ATPase — synthe- lated in the working enzyme. electrochemical potential o 1 o 1 difference of H+ between the sizes cellular ATP from ADP and inorganic phosphate ∆ F and F are both rotary motors two phases is expressed as µH+ (Pi). The energy for ATP synthesis is provided from 1 o = F∆ψ–2.3RT∆pH, where F is downhill H+ (proton) transport along the gradient of ATP synthase is a large protein complex (~500 kDa) the Faraday constant, ∆ψ is the 2 ELECTROCHEMICAL POTENTIAL of protons across membranes with a complicated structure. It is composed of a electric potential difference ∆µ between two phases, R is the gas ( H+). This potential is built by the electron-transfer membrane-embedded portion, Fo(read as ‘ef oh’), constant, T is the absolute chains of respiration or photosynthesis, which pump central and side stalks, and a large headpiece (FIG. 2). temperature and ∆pH is pH γε protons against a gradient (FIG. 1). The central portion (F1 –Foc10–14?) rotates relative to difference between two phases. α β δ Boyer’s admiration for ATP synthase is justified for the surrounding portion (F1 3 3 –Foab2) and, for two reasons. First, ATP supports nearly all the cellular convenience, we will call the former the ‘rotor’ and the activities that require energy. ATP synthesis is the most latter the ‘stator’,although the rotor–stator relation- ∆µ prevalent chemical reaction in the biological world, and ship is relative. When the magnitude of H+ is large, ATP synthase is one of the most ubiquitous, abundant as in functional mitochondria, downhill proton flow proteins on Earth. From Escherichia coli to plants and through Fo causes rotation of the Fo rotor and, hence, γε mammals, this enzyme is one of the most conserved rotation of the -subunits of F1. The rotary motion during evolution, with >60% of the amino-acid residues of the γ alternates the structure of the β-subunit so of the catalytic β-subunit being conserved3–5. Second, that ATP is synthesized. ATP synthase uses physical rotation of its own subunits In the reverse reaction, ATP hydrolysis in F1 γ as a step of catalysis — a novel mechanism, different induces the rotation of and, hence, of the Fo rotor in from that of any other known enzyme. Rotation is not a the reverse direction. This then drives proton pump- favourite motion in living organisms; there is no animal ing. In either case, the side stalk connecting the stator with wheels, no bird with a propeller and no fish with a of Fo and that of F1 prevents them being dragged by screw. On a molecular scale, apart from ATP synthase, the central rotor. It is therefore possible to define the Chemical Resources only bacterial flagella are known as a rotary motor. The ATP synthase as a complex of two motors — an ATP- Laboratory, Tokyo crystal structures of the main part of the ATP synthase driven F1 motor and a proton-driven Fo motor. They Institute of Technology, show, in atomic detail, how the appearance of this tiny are connected by a common rotary shaft and their Nagatsuta 4259, motor made from ~3,500 amino acids is remarkably genuine directions of rotation are opposite. The Yokohama 226-8503, Japan. reminiscent of man-made motors6. motor motions have been visualized for F ,as Correspondence to M.Y. 1 e-mail: The story behind the study of the mechanism of described below and in REF. 7, but this has yet to be [email protected] ATP synthesis contains many dramas (BOX 1), and the done for Fo (REF. 8). NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | SEPTEMBER 2001 | 669 © 2001 Macmillan Magazines Ltd REVIEWS NADH Cytochrome Cytochrome c ATP synthase dehydrogenase bc complex oxidase 1 ADP+Pi ATP NADH NAD+ + O2 H2O H Matrix Q Intermembrane space H+ H+ H+ Cytochrome c Figure 1 | The respiratory chain and ATP synthase. Electrons are transferred from NADH dehydrogenase to cytochrome c oxidase by coenzyme Q (Q), cytochrome bc1 complex and cytochrome c. The established proton gradient across the inner mitochondrial membrane drives the proton flow in ATP synthase that accompanies ATP synthesis. Structures are taken from: 71 72 52 29 cytochrome bc1 complex ; cytochrome c oxidase ; the F1 part of ATP synthase ; and the Fo part of ATP synthase . β Structure of F1 equivalents in the native structure. The -subunit (αβ) γ β ′ In the initial crystal structure of the 3 -portion of equivalent to E takes a ‘half-closed’ (C ) conformation native bovine mitochondrial F1 (termed the ‘native’ (FIG. 3d), and retains Mg-ADP and sulphate (a mimic of structure)6, three α-subunits and three β-subunits are phosphate) at the catalytic site. In the C′-form β, the car- αβ β arranged alternately, forming a cylinder of ( )3 around boxy-terminal domain of the E swings ~23° upwards, the coiled-coil structure of the γ-subunit (FIG. 3a,b).The and the distance between the β-phosphate of ADP and α- and β-subunits have a similar fold, as would be the sulphate would be too long to resynthesize ATP,even expected from their sequence similarity. All of the α- if sulphate were replaced by phosphate. The coiled-coil subunits are bound to the ATP analogue AMP–PNP, region of the γ-subunit twists ~20° in the region sur- αβ and the three subunits adopt very similar conforma- rounded by the ( )3 cylinder and ~10° in the region β αβ tions. The three -subunits, however, are in three protruding from the ( )3 cylinder. β nucleotide-bound states: the first, termed TP, has β Visualizing the rotation of F AMP–PNP in the catalytic site (FIG. 3c); the second ( DP) 1 β γ αβ has ADP; and the third ( E) has no bound nucleotide ATP-driven rotation of the -subunit in the ( )3 cylin- αβ γ (FIG. 3c–e). So, the native structure of F1 looks like a snap- der of F1 has been visualized using the ( )3 -subcom- shot of the working rotary engine, with three reaction plex from a thermophilic bacterium with three micro- chambers representing the moment just after exhaust probes (FIG. 4). With reference to the crystal structure, the β β β γ β and intake ( Ε), ignition ( DP) and compression ( TP) direction of the -rotation is such that one undergoes β β β (BOX 2). The lower part of the slightly bowing, asymmet- a transition in the order TP, DP and E, consistent with ric coiled-coil structure of the γ-subunit is displaced ATP-hydrolysis-driven rotation. At very low concentra- β towards the E, forcing the carboxy-terminal domain of tions of ATP,rotation occurs in discrete 120° steps, each β β this -subunit to swing ~30° downwards. Thus, the E driven by an ATP molecule that arrives at F1. β β adopts the ‘open’ (O) form, whereas TP and DP have Distribution of the dwelling time (a period between one the ‘closed’ (C) form. 120° step and the next) obeys an exponential decrease, 10 A novel crystal structure of bovine F1, with all three confirming that one ATP is consumed per 120° step . catalytic sites occupied by nucleotides, was recently Long actin filaments (FIG. 4) rotate slowly and short reported9. Crystals were grown in Mg-ADP and alu- ones rotate more rapidly, but the torque of rotary – minium fluoride (AlF4 ; a dummy phosphate inhibitor motion, calculated from the rotational velocity of an that is expected to stabilize the conformations of a cat- actin filament and the frictional resistance of water (vis- alytic transition state). In this latest structure — termed cous load), always reached ~40 pN nm–1. The energy – β × (ADP•AlF4 )2F1 — the two s, which are identified as required to produce this magnitude of torque is ~8 β β –20 being equivalent to TP and DP in the native structure 10 J per 120° rotation; the free energy liberated from from their relative positions to the asymmetric γ-sub- one molecule of ATP under the same conditions is ~9 × –20 unit, hold Mg-ADP•AlF4 at their catalytic sites. Their 10 J. So the efficiency of converting the energy of ATP structures are very similar to each other and to their hydrolysis into that of rotation seems to be very high — 670 | SEPTEMBER 2001 | VOLUME 2 www.nature.com/reviews/molcellbio © 2001 Macmillan Magazines Ltd REVIEWS SWITCH II REGION around 90% (REF.10). Lower values (50–80%) have been toring rapid rotation. Rotation is detected by light scat- β The -subunit of F1 has a region reported on the basis of the rotation velocity of nickel tering of a laser beam and recorded by a video with that is topologically equivalent bars attached to the γ-subunit11.

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