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1.
The F1-ATP synthase complex constitutes the catalytic component of F1F0-ATP synthase, the primary ATP synthetic enzyme in the cell. Previous studies indicate that the glacier ice worm, Mesenchytraeus solifugus, maintains unusually high ATP levels that continue to rise as temperatures decline, suggesting that molecular changes within
ice worm F1-ATP synthase subunits may contribute to this energetic anomaly. In this report, we compared ice worm F1-ATP synthase subunits (α, β, γ) with homologues across metazoan phyla (arthropod, chordate, nematode) and among a group of clitellate annelids (Enchytraeus albidus, Enchytraeus buchholzi, Lumbriculus variegatus, Theromyzon tessulatum). Amino acid alignments indicated that ice worm F1-ATP α and F1-ATP β subunits share strong sequence homology with their mesophilic counterparts, respectively, but that ATP γ has diverged more rapidly. Moreover, F1-ATP α and F1-ATP β displayed amino acid compositional changes consistent with trends observed in other cold adapted proteins, while F1-ATP γ diverged in unexpected directions (e.g., gains in size, charged residues). Several ice worm-specific amino acid substitutions
map to positions near the F1-ATP β catalytic site while others occur near subunit contact sites. 相似文献
2.
ATP synthases are motor complexes comprised of F0 and F1 parts that couple the proton gradient across the membrane to the synthesis of ATP by rotary catalysis. Although a great deal of information has been accumulated regarding the structure and function of ATP synthases, their motor functions are not fully understood. For this reason, we performed the alignments and analyses of the protein sequences comprising the core of the ATP synthase motor complex, and examined carefully the locations of the conserved residues in the subunit structures of ATP synthases. A summary of the findings from this bioinformatic study is as follows. First, we found that four conserved regions in the sequence of subunit are clustered into three patches in its structure. The interactions of these conserved patches with the and subunits are likely to be critical for energy coupling and catalytic activity of the ATP synthase. Second, we located a four-residue cluster at the N-terminal domain of mitochondrial OSCP or bacterial (or chloroplast) subunit which may be critical for the binding of these subunits to F1. Third, from the localizations of conserved residues in the subunits comprising the rotors of ATP synthases, we suggest that the conserved interaction site at the interface of subunit c and (mitochondria) or (bacteria and chloroplasts) may be important for connecting the rotor of F1 to the rotor of F0. Finally, we found the sequence of mitochondrial subunit b to be highly conserved, significantly longer than bacterial subunit b, and to contain a shorter dimerization domain than that of the bacterial protein. It is suggested that the different properties of mitochondrial subunit b may be necessary for interaction with other proteins, e.g., the supernumerary subunits. 相似文献
3.
S. Kauffer R. Schmid K. Steffens G. Deckers-Hebestreit K. Altendorf 《Archives of microbiology》1987,148(3):187-192
The ATP synthase complex of Klebsiella pneumoniae (KF1F0) has been purified and characterized. SDS-gel electrophoresis of the purified F1F0 complexes revealed an identical subunit pattern for E. coli (EF1F0) and K. pneumoniae. Antibodies raised against EF1 complex and purified EF0 subunits recognized the corresponding polypeptides of EF1F0 and KF1F0 in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA
9-amino-6-chloro-2-methoxyacridine
- DCCD
N,N-dicyclohexylcarbodiimide
- FITC
fluorescein isothiocyanate
- SDS
sodium dodecyl sulfate
- TTFB
4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole 相似文献
4.
Jui H. Wang 《Journal of bioenergetics and biomembranes》1988,20(4):407-422
Recent studies of chemically modified F1-ATPases have provided new information that requires a revision of our thinking on their catalytic mechanism. One of the subunits in F1-ATPase is distinguishable from the other two both structurally and functionally. The catalytic site and regulatory site of the same subunit are probably sufficiently close to each other, and the interaction between the various catalytic and regulatory sites are probably sufficiently strong to raise the uni-site rate of ATP hydrolysis by several orders of magnitude to that of promoted (multi-site) ATP hydrolysis. Although all three subunits in F1 possess weak uni-site ATPase activity, only one of them () catalyzes promoted ATP hydrolysis. But all three subunits catalyze ATP synthesis driven by the proton flux. Internal rotation of the 33 or 3 moiety relative to the remainder of the F0F1 complex did not occur during oxidative phosphorylation by reconstituted submitochondrial particles. 相似文献
5.
Vik SB 《Journal of bioenergetics and biomembranes》2000,32(5):485-491
The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F1, a water-soluble catalytic sector, and Fo, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed. 相似文献
6.
Roderick A. Capaldi Robert Aggeler Edward P. Gogol Stephan Wilkens 《Journal of bioenergetics and biomembranes》1992,24(5):435-439
The structure of theEscherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the and subunits of the F1 part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 Å at its widest point. The and subunits are interdigitated in side view for around 60 Å of the 90 Å length of the molecule. The F1 narrows and has three-fold symmetry at the end furthest from the F0 part. The F1 is linked to F0 by a stalk approximately 45 Å long and 25–30 Å in diameter. The F0 part is mostly buried in the lipid bilayer. The subunit provides a domain that extends into the central cavity of the F1 part. The and subunits are in a different conformation when ATP+Mg2+ are present in catalytic sites than when ATP+EDTA are present. This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the phosphate of ATP. We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F0 part of the complex. 相似文献
7.
The mitochondrial ATP synthases shares many structural and kinetic properties with bacterial and chloroplast ATP synthases. These enzymes transduce the energy contained in the membrane's electrochemical proton gradients into the energy required for synthesis of high-energy phosphate bonds. The unusual three-fold symmetry of the hydrophilic domain, F1, of all these synthases is striking. Each F1 has three identical subunits and three identical subunits as well as three additional subunits present as single copies. The catalytic site for synthesis is undoubtedly contained in the subunit or an , interface, and thus each enzyme appears to contain three identical catalytic sites. This review summarizes recent isotopic and kinetic evidence in favour of the concept, originally proposed by Boyer and coworkers, that energy from the proton gradient is exerted not directly for the reaction at the catalytic site, but rather to release product from a single catalytic site. A modification of this binding change hypotheses is favored by recent data which suggest that the binding change is due to a positional change in all three subunits relative to the remaining subunits of F1 and F0 and that the vector of rotation is influenced by energy. The positional change, or rotation, appears to be the slow step in the process of catalysis and it is accelerated in all F1F0 ATPases studied by substrate binding and by the proton gradient. However, in the mammalian mitochondrial enzyme, other types of allosteric rate regulation not yet fully elucidated seem important as well. 相似文献
8.
The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 mol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the and subunits of CF1 play key roles in both regulation of activity and proton translocation. 相似文献
9.
Sergio Papa Fzaneo Zanotti Antonio Gaballo 《Journal of bioenergetics and biomembranes》2000,32(4):401-411
The structural and functional connection between the peripheral catalytic F1 sector and theproton-translocating membrane sector F0 of the mitochondrial ATP synthase is reviewed. Theobservations examined show that the N-terminus of subunit , the carboxy-terminal and centralregion of F0I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateralstalk, which connects the peripheries of F1 to F0 and surrounds the central element of thestalk, constituted by subunits and . The ATPase inhibitor protein (IF1) binds at one sideof the F1F0 connection. The carboxy-terminal segment of IF1 apparently binds to OSCP. The42L-58K segment of IF1, which is per se the most active domain of the protein, binds at thesurface of one of the three / pairs of F1, thus preventing the cyclic interconversion of thecatalytic sites required for ATP hydrolysis. 相似文献
10.
L. Mario Amzel Mario A. Bianchet Peter L. Pedersen 《Journal of bioenergetics and biomembranes》1992,24(5):429-433
It has been proposed that during ATP synthesis/hydrolysis F1 ATPases experience a complex pattern of nucleotide binding and release during the catalytic cycle (binding change mechanism). This type of mechanism has implications that can be correlated with the structure of the enzyme. F1-ATPases (stoichiometry 33) are essentially a symmetrical trimer of pairs of the major subunits ( and ); the minor subunits (, and ) are in single copies and interact with the trimer in an asymmetrical fashion. The asymmetry introduced by the minor subunits has important structural and functional consequences: (1) it introduces differences between the potentially equivalent binding and catalytic sites in the major subunits, (2) it restricts the ways in which a binding change mechanism can occur, and (3) it governs the way in which the F1 interacts with the (asymmetrical) F0 sector. 相似文献
11.
Ferruccio Guerrieri Martin Kalous Edmondo Adorisio Nicola Turturro Zdenek Drahota Palmiro Cantatore Giuseppe Santoro 《Journal of bioenergetics and biomembranes》1998,30(3):269-276
In liver mitochondria isolated from hypothyroid rats, the rate of ATP synthesis is lower than in mitochondria from normal rats. Oligomycin-sensitive ATP hydrolase activity and passive proton permeability were significantly lower in submitochondrial particles from hypothyroid rats compared to those isolated from normal rats. In mitochondria from hypothyroid rats, the changes in catalytic activities of F0F1-ATP synthase are accompanied by a decrease in the amount of immunodetected -F1, F01-PVP, and OSCP subunits of the complex. Northern blot hybridization shows a decrease in the relative cytosolic content of mRNA for -F1 subunit in liver of hypothyroid rats. Administration of 3,5,3-triodo-L-thyronine to the hypothyroid rats tends to remedy the functional and structural defects of F0F1-ATP synthase observed in the hypothyroid rats. The results obtained indicate that hypothyroidism leads to a decreased expression of F0F1-ATP synthase complex in liver mitochondria and this contributes to the decrease of the efficiency of oxidative phosphorylation. 相似文献
12.
Roderick A. Capaldi Robert Aggeler Stephan Wilkens Gerhard Grüber 《Journal of bioenergetics and biomembranes》1996,28(5):397-401
Structural changes in theEscherichia coli ATP synthase (ECF1F0) occur as part of catalysis, cooperativity and energy coupling within the complex. The and subunits, two major components of the stalk that links the F1 and F0 parts, are intimately involved in conformational coupling that links catalytic site events in the F1 part with proton pumping through the membrane embedded F0 sector. Movements of the subunit have been observed by electron microscopy, and by cross-linking and fluorescence studies in which reagents are bound to Cys residues introduced at selected sites by mutagenesis. Conformational changes and shifts of the subunit related to changes in nucleotide occupancy of catalytic sites have been followed by similar approaches. 相似文献
13.
Minimal photosynthetic catalytic F1() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF1 and Rhodospirillum rubrum chromatophore RrF1. A CF1-33 hexamer and RrF1-11 dimer, which were purified from the respective F1() complexes, exhibit lower rates and different properties from their parent F1-ATPases. Most interesting is their complete resistance to inhibition by the general F1 inhibitor azide and the specific CF1 inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F1-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F1-ATPases in the 11 dimer is consistant with the view that the dimer contains only a single catalytic site. The 33 hexamer contains however all F1 catalytic sites. Therefore the observation that CF1-33 can bind tentoxin and is stimulated by it suggests that the F1 subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F1-catalytic sites.Abbreviations CF0F1
chloroplast F0F1
- CF1
chloroplast F1
- CF1
chloroplast F1 subunit
- CF1
chloroplast F1 subunit
- CF1()
a complex containing equal amounts of the CF1 and subunits
- MF1
mitochondrial F1
- RrF0F1
Rhodospirillum rubrum F0F1
- RrF1
R. rubrum F1
- RrF1
R. rubrum F1 subunit
- RrF1
R. rubrum F1 subunit
- RrF1()
a complex containing equal amounts of the RrF1 and subunits
- Rubisco
Ribulose-1,5-bisphosphate carboxylase
- TF1
thermophilic bacterium PS3 F1 相似文献
14.
Edgar Vázquez-Contreras Nora Vázquez-Laslop Georges Dreyfus 《Journal of bioenergetics and biomembranes》1995,27(1):109-116
A functional F0F1 ATP synthase that contains the endogenous inhibitor protein (F0F1I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry
14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem.
142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV
max of 1.28mol min–1 mg–1, whereas theV
max of the complex without the inhibitor was 8.3mol min–1 mg–1. In contrast, theK
m
for Mg-ATP of F0F1 I was 148M, comparable to theK
m
value of 142M of the F0F1 complex devoid of IF1. The hydrolytic activity of the F0F1I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min–1 mg–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone. 相似文献
15.
Danièle C. Gautheron Catherine Godinot 《Journal of bioenergetics and biomembranes》1988,20(4):451-468
Monoclonal and polyclonal antibodies directed against peptides of F1-ATPase or F1F0-ATPase synthase provide new and efficient tools to study structure-function relationships and mechanisms of such complex membrane enzymes. This review summarizes the main results obtained using this approach. Antibodies have permitted the determination of the nature of subunits involved in the complex, their stoichiometry, their organization, neighboring interactions, and vectorial distribution within or on either face of the membrane. Moreover, in a few cases, amino acid sequences exposed on a face of the membrane or buried inside the complex have been identified. Antibodies are very useful for detecting the role of each subunit, especially for those subunits which appear to have no direct involvement in the catalytic mechanism. Concerning the mechanisms, the availability of monoclonal antibodies which inhibit (or activate) ATP hydrolysis or ATP synthesis, which modify nucleotide binding or regulation of activities, which detect specific conformations, etc. brings many new ways of understanding the precise functions. The specific recognition by monoclonal antibodies on the subunit of epitopes in the proximity of, or in the catalytic site, gives information on this site. The use of anti- monoclonal antibodies has shown asymmetry of in the complex as already shown for . In addition, the involvement of with respect to nucleotide site cooperativity has been detected. Finally, the formation of F1F0-antibody complexes of various masses, seems to exclude the functional rotation of F1 around F0 during catalysis.Abbreviations IF1
natural protein inhibitor of the ATPase-ATP synthase
- OSCP
oligomycin sensitivity-conferring protein
- DCCD
dicyclohexylcarbodiimide
- SDS-PAGE
sodium dodecylsulfate polyacrylamide gel electrophoreses
- F1
F1-ATPase, coupling factor F1 of ATPase
- F1F0
F1F0-ATP synthase, ATPase-ATP synthase complex 相似文献
16.
We have isolated the F0F1-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM
dodecyl--D-maltoside
- OSCP
oligomycin sensitivity conferring protein
- PMSF
phenyl-methylsulfonylfluoride
- DTT
dithiothreitol
- EDTA
ethylenediaminotetraacetic disodium salt 相似文献
17.
The location of the endogenous inhibitor protein ( IF1) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F1I and intact F1F0I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of –IF1, IF1–IF1, –IF1, and –IF1 cross-linkages in soluble F1I and in native F1F0I complexes. Cross-linking blocked the release of IF1 from its inhibitory site and therefore the activation of F1I and F1F0I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF1 is at a distance 12 Å,to and subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF1 inhibits conformational changes of the catalytic subunits, the inhibitory mechanism of IF1 may involve the interference with rotation of the central stalk. 相似文献
18.
Peter L. Pedersen Young Hee Ko Sangjin Hong 《Journal of bioenergetics and biomembranes》2000,32(4):325-332
This introductory article briefly summarizes how our views about the structural features ofATP synthases (F0F1) have evolved over the past 30 years and also reviews some of our currentviews in the year 2000 about the structures of these remarkably unique enzyme complexes.Suffice it to say that as we approach the end of the first year of this new millinium, we canbe conservatively confident that we have a reasonably good grasp of the overall low-resolutionstructural features of ATP synthases. Electron microscopy techniques, combined with the toolsof biochemistry, molecular biology, and immunology, have played the leading role here byidentifying the headpiece, basepiece, central stalk, side stalk, cap, and in the mitochondrialenzyme, the collar around the central stalk. We can be reasonably confident also that we havea fairly good grasp of much of the high-resolution structural features of both the F1 moietycomprised of fives subunit types (, , , , and ) and parts of the F0 moiety comprised ofeither three (E. coli) or at least ten (mitochondria) subunit types. This information acquiredin several different laboratories, either by X-ray crystallography or NMR spectroscopy, includesdetails about the active site and subunit relationships. Moreover, it is consistent with recentlyreported data that the F1 moiety may be an ATP driven motor, which, during ATP synthesis,is driven in reverse by the electrochemical proton gradient generated by the electron transportchain. The real structural challenges of the future are to acquire at high resolution completeATP synthase complexes representative of different stages of the catalytic cycle during ATPsynthesis and representative also of key regulatory states. 相似文献
19.
Schemes are proposed for coupling sequential opening and closing the three catalytic sites of F1 to rotation of the subunit during ATP synthesis and hydrolysis catalyzed by the FoF1-ATP synthase. A prominent feature of the proposed mechanisms is that the transition state during ATP synthesis is formed when a catalytic site is in the process of closing and that the transition state during ATP hydrolysis is formed when a catalytic site is in the process of opening. The unusual kinetics of formation of Mg-ADP—fluoroaluminate complexes in one or two catalytic sites of nucleotide-depleted MF1 and wild-type and mutant 33 subcomplexes of TF1 are also reviewed. From these considerations, it is concluded that Mg-ADP—fluoroaluminate complexes formed at catalytic sites of isolated F1-ATPases or F1 in membrane-bound FoF1 are ground-state analogs. 相似文献
20.
We have cloned and sequenced the genes atpB and atpE, coding for CF1 subunits and , respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3 to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74–79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits and . A phylogram predicted from F1/CF1- subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae. 相似文献