Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria

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  • Bioenergetic cost of making an adenosinetriphosphate molecule in animal mitochondriaIan N. Watta, Martin G. Montgomerya, Michael J. Runswicka, Andrew G. W. Leslieb,1, and John E. Walkera,1

    aThe Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge, CB2 0XY, United Kingdom; and bThe Medical Research CouncilLaboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, United Kingdom

    Contributed by John E. Walker, August 3, 2010 (sent for review July 9, 2010)

    The catalytic domain of the F-ATPase in mitochondria protrudesinto the matrix of the organelle, and is attached to the membranedomain by central and peripheral stalks. Energy for the synthesis ofATP from ADP and phosphate is provided by the transmembraneproton-motive-force across the inner membrane, generated byrespiration. The proton-motive force is coupled mechanically toATP synthesis by the rotation at about 100 times per second ofthe central stalk and an attached ring of c-subunits in the mem-brane domain. Each c-subunit carries a glutamate exposed aroundthe midpoint of the membrane on the external surface of the ring.The rotation is generated by protonation and deprotonation suc-cessively of each glutamate. Each 360 rotation produces three ATPmolecules, and requires the translocation of one proton per gluta-mate by each c-subunit in the ring. In fungi, eubacteria, and plantchloroplasts, ring sizes of c10c15 subunits have been observed,implying that these enzymes need 3.35 protons to make eachATP, but until now no higher eukaryote has been examined. Asshown here in the structure of the bovine F1-c-ring complex, thec-ring has eight c-subunits. As the sequences of c-subunits are iden-tical throughout almost all vertebrates and are highly conserved ininvertebrates, their F-ATPases probably contain c8-rings also.Therefore, in about 50,000 vertebrate species, and probably inmany or all of the two million invertebrate species, 2.7 protonsare required by the F-ATPase to make each ATP molecule.

    ATP synthase rotational catalysis c-ring structure protons per ATP vertebrates

    Almost all ATP in respiring cells is made by the membranebound enzyme F-ATPase (F-ATP synthase). In the F-ATPasein the inner membranes of mitochondria, the energy of thetransmembrane proton-motive-force, generated by respiration,is coupled mechanically to the synthesis of ATP from ADPand phosphate in its membrane extrinsic catalytic domain by ro-tating the asymmetrical central stalk in a clockwise direction (asviewed from the membrane) at about 100 times per second (14).The spherical catalytic domain, which protrudes into the matrixof the organelle, has three catalytic sites in -subunits at inter-faces with -subunits (5). The rotational torque is resisted bythe peripheral stalk which links the surface of the catalytic do-main to subunit a (ATPase-6) in the membrane domain; togetherthey constitute the stator (6). The asymmetry of the central stalkimposes different conformations on the three catalytic sites. In aground state structure of the catalytic domain, two of them, theDP and the TP sites, have similar but significantly differentclosed conformations. Both bind nucleotides, but catalysis occursat the DP site. The third, or E site, has a different open confor-mation with low nucleotide affinity (5). These three catalyticconformations correspond to tight, loose, and open statesin a binding change mechanism of ATP hydrolysis and synthesis(7). Each 360 rotation of the central stalk takes each catalytic sitethrough these conformations in which substrates bind, and threeATP molecules are made and released. The turning of the rotor isimpelled by protons, driven across the inner membrane into themitochondrial matrix by the transmembrane proton-motive force.The transmembrane pathway for protons in the a-subunit has not

    been defined structurally. This pathway allows protons in the in-termembrane space to access an essential ionized carboxylate of aglutamate residue, midmembrane on the C-terminal -helix ofsubunit c. Once protonated, this carboxylate moves to a morehydrophobic environment by Brownian motion generating a ro-tation of the ring. As succeeding c-subunits become protonated,each neutralized carboxylate reaches an environment in subunit awhere it reionises, releasing the proton into the mitochondrialmatrix (8). According to current models based on structures,the number of translocated protons for generation of each 360rotation is the same as the number of c-subunits in the ring, aseach c-subunit carries a carboxylate involved in protonation anddeprotonation events. In the yeast F-ATPase, the ring has tenc-subunits, and so ten protons are translocated per three ATPmolecules made during a 360 cycle; therefore, the bioenergeticcost to the enzyme is 3.3 protons per ATP (9). However, the c-ringsizes differ between species; c10c15 rings have been found inyeast, eubacterial, and plant chloroplast F-ATPases (1013).Therefore, the bioenergetic cost of these F-ATPases making anATP molecule ranges from 3.35 protons per ATP.

    Until now, the c-ring symmetry and the bioenergetic cost ofmaking an ATP in a mammalian F-ATPase has been unknown.As described here, we have determined the ring size in the struc-ture of the bovine F1-c-ring complex at 3.5 resolution.

    Results and DiscussionIsolation of the Bovine F1-c-ring Complex. The complex was pre-pared from the purified bovine ATP synthase by dissociationof the peripheral stalk, subunit a, and other minor membranesubunits (subunits A6L, e, f, and g) with detergents. The purifiedcomplex consisted of the -, -, -, - and -subunits that consti-tute the catalytic F1-domain plus the membrane protein, sub-unit c. All of these subunits were present in crystals of thecomplex (Fig. S1).

    Structure Determination. The structure of the bovine F1-c-ringcomplex (Fig. 1) was solved by molecular replacement with datato 3.5 resolution. Data processing and statistics are summa-rized in Table 1. The final model contains the following residues:19-510 of the three -subunits, 9-475 of the three -subunits, re-sidues 1-61, 67-96, and 101-272 of the -subunit, residues 15-145of the -subunit, residues 1-47 of the -subunit, and residues 2-73of c-subunits. The c-ring contains eight c-subunits. During refine-

    Author contributions: J.E.W. designed research; I.N.W., M.G.M., M.J.R., and J.E.W.performed research; I.N.W., A.G.W.L., and J.E.W. analyzed data; and J.E.W. wrotethe paper.

    The authors declare no conflict of interest.

    Freely available online through the PNAS open access option.

    Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, (PDB ID code 2xnd).

    See Commentary on page 16755.1To whom correspondence may be addressed. E-mail:

    This article contains supporting information online at PNAS September 28, 2010 vol. 107 no. 39 1682316827







  • ment, an unsuccessful attempt was made to model a c9-ring intothe density (see Fig. S2).

    Structure of the F1-c-ring Complex. In the structure (Fig. 1A), theF1-domain has a ground state structure and the catalytic sitesin subunits TP and DP contain a bound molecule of the ATPanalogue, AMP-PNP, together with Mg2, and the third catalyticsubunit, E, is devoid of nucleotide. Each noncatalytic -subunitalso contains a bound nucleotide and a magnesium ion, but theydo not participate directly in ATP synthesis. A cross-section of thec-ring shows two concentric rings each containing eight regions ofdensity (Fig. 1B). The eight regions in the inner and outer ringcorrespond to the N- and C-terminal -helices of the c-subunit,

    respectively. The latter are significantly longer than the former(52 and 41 , respectively), as in other species. The central holehas a diameter of 47 at the top and bottom, and 42 at theneck in middle of the membrane. The hole is probably occupiedby phospholipids as in other rings from F- and V-type ATPases(10, 14). The ring of eight c-subunits interacts extensively withthe foot of the central stalk via subunits , , and and loop re-gions linking the -helices of each c-subunit in the ring (Fig. S3).The buried surface area in this contact region is 790 2, whereas600 2 was estimated to be buried in the structure of the yeastF1-c10. In both structures, side chains were not built in c-subunits,and so the actual values are higher. Superimposition of the bovineand yeast F1-c-ring structures shows that the rings do not coincide(Fig. S4). The position of the yeast c-ring was influenced bycrystal contacts, and the bovine structure, where the c-ring ismore symmetrically situated, probably provides a more accuraterepresentation of the domain in the intact F-ATPase.

    The c8-ring is the smallest c-ring yet observed amongF-ATPases. Models of c8 and c9-rings were constructed withthe bovine diameter using the structure of a yeast c-subunit froma model of the yeast F1-c10 complex. In both models, there wereserious stereo-chemical clashes between the side chains of resi-dues Ile-13, Leu-19, and Ile-23 with the equivalent side chainsof an adjacent protomer in the neck of the hour-glass shapedc-ring. However, in the bovine c-subunit, these residues are allalanines. In the bovine c8-ring, there are no side chain clashesin the neck of the ring, and in a model of a c9-ring using thebovine structure and sequence, they were greatly reduced relativeto the yeast c9- and c8-rings. Replacement of these alanine resi-dues with amino acids with larger side chains, such as valine, leadsto side chain clashes and destabilizes the ring. Replacement ofthe alanines by glycines abolishes hydrophobic packing interac-tions that contribute to the rings stability. Only in one inverte-brate species, the tube-worm, Hydroides elegans, is alanine-13evidently replaced by cysteine. Therefore, the presence of alanineresidues at positions 13, 19, and 23 (bovine numbering) appearsto be essential for the formation of the c8-ring.

    Mosaic Structure of Bovine ATP Synthase.The structure of the bovineF1-c-ring provides an important contribution to the overall struc-ture of bovine ATP synthase. In Fig. 2, a mosaic structure hasbeen built by docking high resolution component structures intoa structure of the intact enzyme determined by electron cryomi-croscopy at 32 resolution (pale gray outline in Fig. 2) (15). Themembrane extrinsic part of the structure is derived from a struc-ture of bovine F1-ATPase with most of the peripheral stalkattached to it (16), augmented by information from a structureof an overlapping fragment of the peripheral stalk (17). Manystructures have been determined of bovine F1-ATPase inhibitedin various ways. The most accurate at 1.9 resolution is a struc-ture representing the ground state in the catalytic cycle of ATPhydrolysis in which the enzyme was inhibited with the nonhydro-lysable ATP analogue, AMP-PNP (18). The empty gray region onthe right of Fig. 2 represents the part of the structure in themembrane domain of the enzyme where detailed structural in-formation is lacking for subunit a (ATPase-6), which sits close tothe surface of the c-ring and provides a transmembrane path forprotons. Also, it contains the membrane intrinsic region of sub-unit b (two transmembrane -helices), and for subunits A6L, e, f,and g (each with a single transmembrane -helix), which are notinvolved directly in the synthesis of ATP. It is likely that, as in thestructure of the V-ATPase from Thermus thermophilus at 16 resolution, the gray area in the membrane domain will containan annular belt of detergent (19).

    The bovine c-subunit has an unusual feature as the -aminogroup of Lys-43, which is at the beginning of the C-terminal-helix, is completely trimethylated (20). The trimethylated sidechain, terminated by a quaternary amino group (which is similar

    Fig. 1. The structure of the bovine F1-c-ring complex. Part A. The subunits inthe F1-catalytic domain (above) and the c-ring (below) are shown in ribbonrepresentation. The membrane extrinsic region consists of the sphericalcatalytic domain, made of three - and three -subunits (red and yellow,respectively), the central stalk (subunits , , and , blue, purple, and green,respectively), The central stalk and the c-ring (brown) together constitute therotor. Each of the C-terminal -helices in the c-ring has a glutamate at residue58, at the midpoint of the lipid bilayer. Part B. Cross-section of the c-ringtaken at the midpoint of the -helices. Above is shown the electron densitywith the eight N-terminal -helices in the inner ring, and the eight C-terminal-helices in the outer ring, and below the structural interpretation of thedensity.

    Table 1. Data collection and refinement statistics for the F1-c-ringcomplex from bovine ATP synthase

    Space group P212121Unit cell dimensions a, b, c, 155.7, 157.1, 247.3Resolution range 50.483.50No. unique reflections 72,959Multiplicity 3.6 (3.5)*Completeness, % 99.8 (99.6)*Rmerge 0.263 (0.817)*hIIi 3.7 (1.6)*B factor, from Wilson plot, 2 72.64R factor, % 26.13Free R factor, % 30.40rmsd of bonds, 0.006rmsd of angles, 0.973

    *Values in parentheses are for the highest resolution bin (3.503.69 )The free R-factor was calculated for 3,855 reflections excluded fromrefinement.

    16824 Watt et al.

  • to choline), is exposed to the phospholipid bilayer in the head-group region. In this position, this side chain would clash withthe head groups of phospholipids and would probably impedetheir binding to the ring. Cardiolipin is a characteristic and abun-dant lipid of the...


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