Structure of bovine mitochondrial F(1)-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis

The crystal structure of a novel aluminium fluoride inhibited form of bovine mitochondrial F(1)-ATPase has been determined at 2 A resolution. In contrast to all previously determined structures of the bovine enzyme, all three catalytic sites are occupied by nucleotide. The subunit that did not bind nucleotide in previous structures binds ADP and sulfate (mimicking phosphate), and adopts a "half-closed" conformation. This structure probably represents the posthydrolysis, pre-product release step on the catalytic pathway. A catalytic scheme for hydrolysis (and synthesis) at physiological rates and a mechanism for the ATP-driven rotation of the gamma subunit are proposed based on the crystal structures of the bovine enzyme.     

Structural model of F1-ATPase and the implications for rotary catalysis

The crystal structure of bovine mitochondrial F1-ATPase is described. Several features of the structure are consistent with the binding change mechanism of catalysis, in which binding of substrates induces conformational changes that result in a high degree of cooperativity between the three catalytic sites. Furthermore, the structure also suggests that catalysis is accompanied by a physical rotation of the centrally placed gamma-subunit relative to the approximately spherical alpha3beta3 subassembly.     

Principal role of the arginine finger in rotary catalysis of F1-ATPase

F(1)-ATPase (F(1)) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α(3)β(3) stator ring. The 3 catalytic sites of F(1) reside on the interface of the α and β subunits of the α(3)β(3) ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins. However, the principal role of the arginine finger of F(1) remains controversial. We studied the role of the arginine finger by analyzing the rotation of a mutant F(1) with a lysine substitution of the arginine finger. The mutant showed a 350-fold longer catalytic pause than the wild-type; this pause was further lengthened by the slowly hydrolyzed ATP analog ATPγS. On the other hand, the mutant F(1) showed highly unidirectional rotation with a coupling ratio of 3 ATPs/turn, the same as wild-type, suggesting that cooperative torque generation by the 3 β subunits was not impaired. The hybrid F(1) carrying a single copy of the α mutant revealed that the reaction step slowed by the mutation occurs at +200° from the binding angle of the mutant subunit. Thus, the principal role of the arginine finger is not to mediate cooperativity among the catalytic sites, but to enhance the rate of the ATP cleavage by stabilizing the transition state of ATP hydrolysis. Lysine substitution also caused frequent pauses because of severe ADP inhibition, and a slight decrease in ATP-binding rate.     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.     

The structure of bovine F1-ATPase in complex with its regulatory protein IF1

In mitochondria, the hydrolytic activity of ATP synthase is prevented by an inhibitor protein, IF1. The active bovine protein (84 amino acids) is an alpha-helical dimer with monomers associated via an antiparallel alpha-helical coiled coil composed of residues 49-81. The N-terminal inhibitory sequences in the active dimer bind to two F1-ATPases in the presence of ATP. In the crystal structure of the F1-IF1 complex at 2.8 A resolution, residues 1-37 of IF1 bind in the alpha(DP)-beta(DP) interface of F1-ATPase, and also contact the central gamma subunit. The inhibitor opens the catalytic interface between the alpha(DP) and beta(DP) subunits relative to previous structures. The presence of ATP in the catalytic site of the beta(DP) subunit implies that the inhibited state represents a pre-hydrolysis step on the catalytic pathway of the enzyme.     

Guanidine-induced dissociation of mitochondrial F1-ATPase

The effect of guanidine hydrochloride on ATPase activity, gel filtration, turbidity, and the fluorescence emission intensity of mitochondrial F1-ATPase was examined. Purified F1 from bovine heart mitochondria was slowly inactivated at low denaturant concentration, and inactivation was associated with delta and epsilon subunit dissociation. delta and epsilon subunits were bound together to form a stable and soluble heterodimer. In parallel, appearance of turbidity was observed. This was caused by the formation of alpha3beta3gamma non-covalent aggregates, as analyzed by SDS-PAGE. Short periods of exposition of the F1 complex to high concentrations of guanidine hydrochloride (0.8-3 M) again induced deltaepsilon dissociation as a heterodimer and the formation of an inactive alpha3beta3gamma subcomplex. This eventually dissociated progressively into single subunits caused by partial unfolding, as evidenced through changes of the protein intrinsic fluorescence emission. Our results suggest that the delta and epsilon subunits are loosely bound to alpha3beta3gamma , and play an important role in determining structural stability to isolated mitochondrial F1-ATPase.     

Cross-linking study of the quaternary fine structure of mitochondrial F1-ATPase

A method has been developed for exploring the quaternary fine structure of oligomeric proteins by crosslinking studies and applied to bovine heart mitochondrial F1-ATPase. The F1 was first labeled with 1-fluoro-2,4-dinitro-[14C]benzene, subsequently reduced with sodium hydrosulfite, and finally cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Gel electrophoresis in the chemically modified protein in the presence of sodium dodecyl sulfate and mercaptoethanol showed the existence of a 105-115-kilodalton molecular species in addition to the five monomeric subunits of F1. This cross-linked species could be alpha 2, alpha beta, or beta 2. Isolation of the cross-linked species and titration with 5,5'-dithiobis-(2-nitrobenzoic acid) showed the absence of sulfhydryl group. Therefore, the cross-linked species must be the dimer beta 2. After digestion of the purified beta 2 with pepsin, a single radioactive peptide was isolated. Determination of the amino acid sequence of this peptide and comparison of its radioactivity with the total radioactivity on beta-subunits show that it was formed exclusively by cross-linking Lys162 of one beta-subunit with Glu199 of another beta-subunit. The observation that two beta-subunits can be cross-linked by a rigid phenylenediamine bridge of 5.7- or 4.3-A length is difficult to reconcile with the widely assumed structure of F1 with the alpha- and beta-subunits occupying alternate corners of a planar hexagon, but is consistent with the structure in which a triangular set of three beta-subunits sits above a triangular set of three alpha-subunits in a staggered conformation.     

Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.     

1H-NMR studies on nucleotide binding to the catalytic sites of bovine mitochondrial F1-ATPase

The conformation of adenine nucleotides bound to bovine mitochondrial F1-ATPase was investigated using transfer nuclear Overhauser enhancement measurements. It is shown that all nucleotides investigated adopt a predominantly anti conformation when bound to the catalytic sites. Furthermore, the experiment suggests that 8-azido-ADP and 8-azido-ATP, which are predominantly in the syn conformation in solution, are in the anti conformation when bound to F1 catalytic sites.     

An electron microscopic approach to the quaternary structure of mitochondrial F1-ATPase

The three-dimensional structure of F1-ATPase from beef heart mitochondria was investigated by electron microscopic techniques. The presence of high concentrations of nucleotides is essential for preservation of the quaternary structure. When investigated under such conditions, monodisperse F1-ATPase could not be distinguished from the membrane-bound enzyme. At low resolution, the particle shape resembles an oblate ellipsoid of revolution with an axial ratio of about 2:1. From several lines of evidence (including field micrographs at higher magnifications, Markham rotational analysis, and tilting experiments), two conclusions may be drawn concerning the three-dimensional fine structure of F1-ATPase. 1. At the periphery of the molecule, six globular protein masses are orientated in a way similar to the chair conformation of cyclohexane. This array is interpreted to be made up of an alternating sequence of alpha and beta subunits. 2. Part of the central space is occupied by a seventh protein mass, protrusions of which are likely to be in contact with some of the outer subunits. A gamma subunit is supposed to be constituent part of this central protein mass. As a consequence, this model favours a stoichiometry of alpha 3 beta 3 gamma for the large subunits of beef heart F1-ATPase.     

Diffusion limited component of mitochondrial F1-ATPase

• 1.1. The possibility that the rate of ATP hydrolysis by F₁-ATPase approaches the diffusion-controlled limits was investigated by measuring the values of k_cat and k_l (k_cat/K_m) as a function of increasing viscosity.
• 2.2. The values of k_cat/K_m decrease significantly with increasing viscosity; further such decrease was lower when F₁-ATPase hydrolyzed poor substrate such as Ca- and Mg-ITP or when the hydrolysis rates were measured at temperatures below 20°C.
• 3.3. Viscosity also decreases _cat, but only at high concentrations of viscosogenic agents.
• 4.4. These results suggest that ATP hydrolysis is at least partly diffusion-controlled, although a general non-specific perturbation in the enzyme structure is also effected by viscosity.

     

Conformational role of the divalent metal in bovine heart mitochondrial F1-ATPase: an electron spin echo envelope modulation study

The catalytic sites of beef heart mitochondrial F1-ATPase were studied by electron spin echo envelope modulation (ESEEM) spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II), maintaining the enzyme catalytic activity. F1-ATPase was purified from beef heart mitochondria. A protein still containing three endogenous nucleotides, named MF1(1,2), is obtained under milder conditions, whereas a harsher treatment gives a fully depleted F1, named MF1(0,0). Several samples were prepared, loading MF1(0,0) or MF1(1,2) with Mn(II) or MnIIADP in both substoichiometric and excess amounts. When MF1(1,2) is loaded with Mn(II) in a 1:0.8 ratio, the FT-ESEEM spectrum shows evidence of a nitrogen interacting with the metal, while this interaction is not present in MF1(0,0) + Mn(II) in a 1:0.8 ratio. However, when MF1(0,0) is loaded with 2.4 Mn(II), the FT-ESEEM spectrum shows a metal-nitrogen interaction resembling that present in MF1(1,2) + Mn(II) in a 1:0.8 ratio. These results strongly support the role of the metal alone in shaping and structuring the catalytic sites of the enzyme. When substoichiometric ADP is added to MF1(1,2) preloaded with 0.8 equiv of Mn(II), the ESEEM spectra show evidence of a phosphorus nucleus coupled to the metal, indicating that the nucleotide phosphate binding to Mn(II) occurs in a catalytic site. Generally, 14N coordination to the metal is clearly identified in the ESEEM spectra of all the samples containing more than one metal equivalent. One point of note is that the relevant nitrogen-containing ligand(s), responsible for the signals in the ESEEM spectra, has not yet been identified in the available X-ray structures.     

Rotary catalysis of the stator ring of F1-ATPase

F₁-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α₃β₃ ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a “dictator” completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F₁-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Rat liver mitochondrial F1-ATPase, an FAD containing ferroprotein.

F1-ATPase isolated from rat liver mitochondria has been found to contain approximately 1 mole of FAD and 6 g atoms of nonheme iron per mole of enzyme.     

Electron microscopy of beef heart mitochondrial F1-ATPase

The quaternary structure of isolated and membrane-bound F1-ATPase (submitochondrial particles) has been studied by electron microscopy. A model of the molecule has been proposed: six protein masses are arranged in two layers approximately at the vertices of a triangular antiprism. Computer averaging of the images showed that the frontal view of the molecule can be approximately characterized by mirror plane symmetry.     

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F₁F₀-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F₁F₀-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 ± 3.0 sec in preconditioned vs. 83.4 ± 4.4 sec in controls, p 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.     

Oxidative phosphorylation in a hybrid system containing bovine heart membranes and pea mitochondrial F1-ATPase

Purified pea mitochondrial F1-ATPase reconstituted oxidative phosphorylation in both partially and completely F1-depleted bovine heart mitochondrial membranes. The isolated plant enzyme exhibited high rates of ATP synthesis when combined with bovine heart membranes, suggesting great evolutionary conservation of the ATP synthase complex in mitochondria.     

The structure of the central stalk in bovine F(1)-ATPase at 2.4 A resolution

The central stalk in ATP synthase, made of gamma, delta and epsilon subunits in the mitochondrial enzyme, is the key rotary element in the enzyme's catalytic mechanism. The gamma subunit penetrates the catalytic (alpha beta)(3) domain and protrudes beneath it, interacting with a ring of c subunits in the membrane that drives rotation of the stalk during ATP synthesis. In other crystals of F(1)-ATPase, the protrusion was disordered, but with crystals of F(1)-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed. The delta and epsilon subunits interact with a Rossmann fold in the gamma subunit, forming a foot. In ATP synthase, this foot interacts with the c-ring and couples the transmembrane proton motive force to catalysis in the (alpha beta)(3) domain.