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1.
We have proposed a model for the oligomeric c-rotor of the Fo sector of ATP synthase and its interaction with subunit a during H+-transport driven rotation. The model is based upon the solution structure of monomeric subunit c, determined by NMR, and an extensive series of cross-linking distance constraints between c subunits and between subunits c and a. To explain the complete set of cross-linking data, we have suggested that the second transmembrane helix rotates during its interaction with subunit a in the course of the H+-translocation cycle. The H+-transport coupled rotation of this helix is proposed to drive the stepwise movement of the c-oligomeric rotor. The model is testable and provides a useful framework for addressing questions raised by other experiments. 相似文献
2.
ATPases with unusual membrane-embedded rotor subunits were found in both F1F0 and A1A0 ATP synthases. The rotor subunit c of A1A0 ATPases is, in most cases, similar to subunit c from F0. Surprisingly, multiplied c subunits with four, six, or even 26 transmembrane spans have been found in some archaea and these multiplication events were
sometimes accompanied by loss of the ion-translocating group. Nevertheless, these enzymes are still active as ATP synthases.
A duplicated c subunit with only one ion-translocating group was found along with “normal” F0 c subunits in the Na+ F1F0 ATP synthase of the bacterium Acetobacterium woodii. These extraordinary features and exceptional structural and functional variability in the rotor of ATP synthases may have
arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life. 相似文献
3.
In Propionigenium modestum, ATP is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of Na+ ions. The P. modestum ATP synthase is a clear member of the family of F-type ATP synthases and the only major distinction is an extension of the coupling ion specificity to H+, Li+, or Na+, depending on the conditions. The use of Na+ as a coupling ion offers unique experimental options to decipher the ion-translocation mechanism and the osmotic and mechanical behavior of the enzyme. The single a subunit and the oligomer of c subunits are part of the stator and rotor, respectively, and operate together in the ion-translocation mechanism. During ATP synthesis, Na+ diffuses from the periplasm through the a subunit channel onto the Na+ binding site on a c subunit. From there it dissociates into the cytoplasm after the site has rotated out of the interface with subunit a. In the absence of a membrane potential, the rotor performs Brownian motions into either direction and Na+ ions are exchanged between the two compartments separated by the membrane. Upon applying voltage, however, the direction of Na+ flux and of rotation is biased by the potential. The motor generates torque to drive the rotation of the subunit, thereby releasing tightly bound ATP from catalytic sites in F1. Hence, the membrane potential plays a pivotal role in the torque-generating mechanism. This is corroborated by the fact that for ATP synthesis, at physiological rates, the membrane potential is indispensable. We propose a catalytic mechanism for torque generation by the Fo motor that is in accord with all experimental data and is in quantitative agreement with the requirement for ATP synthesis. 相似文献
4.
H+-transporting, F1Fo-type ATP synthases utilize a transmembrane H+ potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating β subunits of the extramembranous F1 sector of the enzyme, synthesis being driven by rotation of the γ subunit in the center of the F1 molecule between the alternating catalytic sites . The H+ electrochemical potential is thought to drive γ subunit rotation by first coupling H+ transport to rotation of an oligomeric rotor of c subunits within the transmembrane Fo sector. The γ subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the γ and ε subunits of F1. In this essay we will review recent studies on the Escherichia coli Fo sector. The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp61 centered in the second transmembrane helix (TMH). A model for the structural organization of the c10 oligomer in Fo was deduced from extensive cross-linking studies and by molecular modeling. The model indicates that the H+-carrying carboxyl of subunit c is occluded between neighboring subunits of the c10 oligomer and that two c subunits pack in a “front-to-back” manner to form the H+ (cation) binding site. In order for protons to gain access to Asp61 during the protonation/deprotonation cycle, we propose that the outer, Asp61-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp61 protonation/deprotonation drives the rotation of the c-ring. The NMR structures of wild-type subunit c differs according to the protonation state of Asp61. The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional mutant proteins in which critical residues have been switched between TMH-1 and TMH-2. The structural information is considered in the context of the possible mechanism of rotary movement of the c10 oligomer during coupled synthesis of ATP. 相似文献
5.
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
6.
Jörg-Christian Greie Gabriele Deckers-Hebestreit Karlheinz Altendorf 《Journal of bioenergetics and biomembranes》2000,32(4):357-364
Membrane-bound ATP synthases (F1F0) catalyze the synthesis of ATP via a rotary catalyticmechanism utilizing the energy of an electrochemical ion gradient. The transmembrane potentialis supposed to propel rotation of a subunit c ring of F0 together with subunits and of F1,hereby forming the rotor part of the enzyme, whereas the remainder of the F1F0 complexfunctions as a stator for compensation of the torque generated during rotation. This reviewfocuses on our recent work on the stator part of the F0 complex, e.g., subunits a and b. Usingepitope insertion and antibody binding, subunit a was shown to comprise six transmembranehelixes with both the N- and C-terminus oriented toward the cytoplasm. By use of circulardichroism (CD) spectroscopy, the secondary structure of subunit b incorporated intoproteoliposomes was determined to be 80% -helical together with 14% turn conformation, providingflexibility to the second stalk. Reconstituted subunit b together with isolated ac subcomplexwas shown to be active in proton translocation and functional F1 binding revealing the nativeconformation of the polypeptide chain. Chemical crosslinking in everted membrane vesiclesled to the formation of subunit b homodimers around residues bQ37 to bL65, whereas bA32Ccould be crosslinked to subunit a, indicating a close proximity of subunits a and b near themembrane. Further evidence for the proposed direct interaction between subunits a and b wasobtained by purification of a stable ab
2 subcomplex via affinity chromatography using Histags fused to subunit a or b. This ab
2 subcomplex was shown to be active in proton translocationand F1 binding, when coreconstituted with subunit c. Consequences of crosslink formationand subunit interaction within the F1F0 complex are discussed. 相似文献
7.
Robert H. Fillingame 《Journal of bioenergetics and biomembranes》1992,24(5):485-491
The F0 sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F1 sector, couples proton transport to ATP synthesis. The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on theEscherichia coli F0 sector is reviewed here. TheE. coli F0 is composed of three types of subunits (a, b, andc) and current information on their folding and organization in F0 is reviewed. The structure of purified subunitc in chloroform-methanol-H2O resembles that in native F0, and progress in determining the structure by NMR methods is reviewed. Genetic experiments suggest that the two helices of subunitc must interact as a functional unit around an essential carboxyl group as protons are transported. In addition, a unique class of suppressor mutations identify a transmembrane helix of subunita that is proposed to interact with the bihelical unit of subunitc during proton transport. The role of multiple units of subunitc in coupling proton translocation to ATP synthesis is considered. The special roles of Asp61 of subunitc and Arg210 of subunita in proton translocation are also discussed. 相似文献
8.
The mitochondrial ATP synthase is a membrane protein complex which couples the proton gradient across the mitochondrial inner membrane to the synthesis of ATP from ADP + Pi. The complex is composed of essential subunits for its motor functions and supernumerary subunits, the roles of which remain to be elucidated. Subunits g and A6L are supernumerary subunits, and the specific roles of these subunits are still matters of debate. To gain insight into the functions of these two subunits, we carried out the alignment and the homolog search of the protein sequences of the subunits and found the following features: Subunit g appears to have isoforms in animals, and the transmembrane domain of the animal subunit g contains a completely conserved acidic residue in the middle of a helix on the conserved side of the transmembrane helix. This finding implicates the conserved acidic residue as important for the function of subunit g. The alignment of A6L protein sequences shows a conserved aromatic residue at the N-terminal domain with which the N-terminal MPQL sequence comprises a unique MPQLX4Ar motif that can signify the protein A6L. The conserved aromatic residue may also be important for the function of A6L. 相似文献
9.
Asha?Manikkoth Balakrishna Holger Seelert Sven-Hendric Marx Norbert?A. Dencher Gerhard Grüber 《Bioscience reports》2014,34(2)
In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector.Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å. 相似文献
10.
Richter ML Samra HS He F Giessel AJ Kuczera KK 《Journal of bioenergetics and biomembranes》2005,37(6):467-473
The chloroplast F0F1-ATP synthase-ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical
proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the γ subunit
of the chloroplast F1 enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthases
of higher plants and its physiological significance lies in preventing nonproductive depletion of essential ATP pools in the
dark. The three-dimensional structure of the chloroplast F1 gamma subunit has not yet been solved. To examine the mechanism of dithiol regulation, a model of the chloroplast gamma subunit
was obtained through segmental homology modeling based on the known structures of the mitochondrial and bacterial γ subunits,
together with de novo construction of the unknown regulatory domain. The model has provided considerable insight into how the dithiol might modulate
catalytic function. This has, in turn, suggested a mechanism by which rotation of subunits in F0, the transmembrane proton channel portion of the enzyme, can be coupled, via the ε subunit, to rotation of the γ subunit
of F1 to achieve the 120° (or 90°+30°) stepping action that is characteristic of F1 γ subunit rotation. 相似文献
11.
Subunit a plays a key role in promoting H+ transport-coupled rotary motion of the subunit c ring in F1Fo ATP synthase. H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of Fo subunit c. H+ are thought to reach cAsp61 via aqueous half-channels formed by TMHs 2–5 of subunit a. Movements of TMH4 and TMH5 have been proposed to facilitate protonation of cAsp61 from a half channel centered in a four helix bundle at the periplasmic side of subunit a. The possible necessity of these proposed TMH movements was investigated by assaying ATP driven H+ pumping function before and after cross-linking paired Cys substitutions at the center of TMHs within subunit a. The cross-linking of the Cys pairs aG218C/I248C in TMH4 and TMH5, and aL120C/H245C in TMH2 and TMH5, inhibited H+ pumping by 85–90%. H+ pumping function was largely unaffected by modification of the same Cys residues in the absence of cross-link formation. The inhibition is consistent with the proposed requirement for TMH movements during the gating of periplasmic H+ access to cAsp61. The cytoplasmic loops of subunit a have been implicated in gating H+ release to the cytoplasm, and previous cross-linking experiments suggest that the chemically reactive regions of the loops may pack as a single domain. Here we show that Cys substitutions in these domains can be cross-linked with retention of function and conclude that these domains need not undergo large conformational changes during enzyme function. 相似文献
12.
P. Dimroth 《Antonie van Leeuwenhoek》1994,65(4):381-395
For many bacteria Na+ bioenergetics is important as a link between exergonic and endergonic reactions in the membrane. This article focusses on two primary Na+ pumps in bacteria, the Na+-translocating oxaloacetate decarboxylase ofKlebsiella pneumoniae and the Na+-translocating F1F0 ATPase ofPropionigenium modestum. Oxaloacetate decarboxylase is an essential enzyme of the citrate fermentation pathway and has the additional function to conserve the free energy of decarboxylation by conversion into a Na+ gradient. Oxaloacetate decarboxylase is composed of three different subunits and the related methylmalonyl-CoA decarboxylase consists of five different subunits. The genes encoding these enzymes have been cloned and sequenced. Remarkable are large areas of complete sequence identity in the integral membrane-bound -subunits including two conserved aspartates that may be important for Na+ translocation. The coupling ratio of the decarboxylase Na+ pumps depended on
and decreased from two to zero Na+ uptake per decarboxylation event as
increased from zero to the steady state level.InP. modestum,
is generated in the course of succinate fermentation to propionate and CO2. This
is used by a unique Na+-translocating F1F0 ATPase for ATP synthesis. The enzyme is related to H+-translocating F1F0 ATPases. The F0 part is entirely responsible for the coupling of ion specificity. A hybrid ATPase formed by in vivo complementation of anEscherichia coli deletion mutant was completely functional as a Na+-ATP synthase conferring theE. coli strain the ability of Na+-dependent growth on succinate. The hybrid consisted of subunits a, c, b, and part of fromP. modestum and of the remaining subunits fromE. coli. Studies on Na+ translocation through the F0 part of theP. modestum ATPase revealed typical transporter-like properties. Sodium ions specifically protected the ATPase from the modification of glutamate-65 in subunit c by dicyclohexylcarbodiimide in a pH-dependent manner indicating that the Na+ binding site is at this highly conserved acidic amino acid residue of subunit c within the middle of the membrane. 相似文献
13.
Summary The atp operon from the extreme alkaliphile Bacillus firmus OF4 was cloned and sequenced, and shown to contain genes for the eight structural subunits of the ATP synthase, preceded by a ninth gene predicted to encode a 14 kDa hydrophobic protein. The arrangement of genes is identical to that of the atp operons from Escherichia coli, Bacillus megaterium, and thermophilic Bacillus PS3. The deduced amino acid sequences of the subunits of the enzyme are also similar to their homologs in other ATP synthases, except for several unusual substitutions, particularly in the a and c subunits. These substitutions are in domains that have been implicated in the mechanism of proton translocation through F0-ATPase, and therefore could contribute to the gating properties of the alkaliphile ATP synthase or its capacity for proton capture. 相似文献
14.
Brian D. Cain 《Journal of bioenergetics and biomembranes》2000,32(4):365-371
The a and b subunits constitute the stator elements in the F0 sector of F1F0-ATP synthase.Both subunits have been difficult to study by physical means, so most of the information onstructure and function relationships in the a and b subunits has been obtained using mutagenesisin combination with biochemical methods. These approaches were used to demonstrate thatthe a subunit in association with the ring of c subunits houses the proton channel throughF1F0-ATP synthase. The map of the amino acids contributing to the proton channel is probablycomplete. The two b subunits dimerize, forming an extended flexible unit in the peripheralstalk linking the F1 and F0 sectors. The unique characteristics of specific amino acid substitutionsaffecting the a and b subunits suggested differential effects on rotation during F1F0-ATPaseactivity. 相似文献
15.
Susan M. Howitt Andrew J. W. Rodgers Lyndall P. Hatch Frank Gibson Graeme B. Cox 《Journal of bioenergetics and biomembranes》1996,28(5):415-420
F0F1-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F0F1-ATPase. Because of the subunit stoichiometry and the involvement of both thea- andc-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed. 相似文献
16.
Makoto Araki Kazuya Hoshi Masasuke Fujiwara Yuka Sasaki Hideo Yonezawa Hidenobu Senpuku Atsuko Iwamoto-Kihara Masatomo Maeda 《Journal of bacteriology》2013,195(21):4873-4878
The c subunit of Streptococcus mutans ATP synthase (FoF1) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid FoF1 is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F1 bound to the hybrid Fo with the S. mutans
c subunit showed N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli FoF1. Thus, the S. mutans
c subunit assembled into a functional Fo together with the E. coli
a and b subunits, forming a normal F1 binding site. Although the H+ pathway should be functional, as was suggested by the growth on minimum succinate medium, ATP-driven H+ transport could not be detected with inverted membrane vesicles in vitro. This observation is partly explained by the presence of an acidic residue (Glu-20) in the first transmembrane helix of the S. mutans
c subunit, since the site-directed mutant carrying Gln-20 partly recovered the ATP-driven H+ transport. Since S. mutans is recognized to be a primary etiological agent of human dental caries and is one cause of bacterial endocarditis, our system that expresses hybrid Fo with the S. mutans
c subunit would be helpful to find antibiotics and chemicals specifically directed to S. mutans. 相似文献
17.
Rotary catalysis in F1F0 ATP synthase is powered by proton translocation through the membrane-embedded F0 sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag+ or Cd2+ on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag+ and Cd2+ sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag+ or Cd2+ chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F1 from F0 with release of F1 from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F1 after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H+ translocation through F0 as assayed with F0 reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H+ translocation to the cytoplasm. 相似文献
18.
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
19.
Bhatt D Cole SP Grabar TB Claggett SB Cain BD 《Journal of bioenergetics and biomembranes》2005,37(2):67-74
The peripheral stalk of F1F0 ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F0 to the top of the F1 sector. The stalk serves as the stator necessary for holding F1 against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F1. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F1F0 complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F1F0 ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F1F0 complex by a normal b subunit. 相似文献
20.
A homodimer of b subunits constitutes the peripheral stalk linking the F1 and F0 sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F1F0 ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F1F0 ATPase and no detectable ATP-driven proton pumping activity. Expression of the b
N2A,T6A,Q10A
subunit was also oxidative phosphorylation deficient, but the b
N2A,T6A,Q10A
protein was incorporated into an F1F0 complex. Single amino acid substitutions had minimal reductions in F1F0 ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F0, and that these interactions are strongest on the periplasmic side of the bilayer. 相似文献