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1.
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   

2.
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   

3.
E. coli F1-ATPase has been studied mainly by the genetic approach. Mutations in either the or subunit modified the kinetics of multisite and uni-site hydrolysis of ATP. The mechanism of F1-ATPase and the essential amino acid residues of subunits are discussed.Abbreviations used: Pi, inorganic phosphate; DCCD, dicyclohexylcarbodiimide.  相似文献   

4.
The mitochondrial ATP synthase is a molecular motor that drives the phosphorylation ofADP to ATP. The yeast mitochondrial ATP synthase is composed of at least 19 differentpeptides, which comprise the F1 catalytic domain, the F0 proton pore, and two stalks, oneof which is thought to act as a stator to link and hold F1 to F0, and the other as a rotor.Genetic studies using yeast Saccharomyces cerevisiae have suggested the hypothesis thatthe yeast mitochondrial ATP synthase can be assembled in the absence of 1, and even 2, ofthe polypeptides that are thought to comprise the rotor. However, the enzyme complexassembled in the absence of the rotor is thought to be uncoupled, allowing protons to freelyflow through F0 into the mitochondrial matrix. Left uncontrolled, this is a lethal process andthe cell must eliminate this leak if it is to survive. In yeast, the cell is thought to lose ordelete its mitochondrial DNA (the petite mutation) thereby eliminating the genes encodingessential components of F0. Recent biochemical studies in yeast, and prior studies in E. coli,have provided support for the assembly of a partial ATP synthase in which the ATP synthaseis no longer coupled to proton translocation.  相似文献   

5.
Proton-translocating ATP synthase (FoF1) synthesizes ATP from ADP and phosphate, coupled with an electrochemical proton gradient across the biological membrane. It has been established that the rotation of a subunit assembly is an essential feature of the enzyme mechanism and that FoF1 can be regarded as a molecular motor. Thus, experimentally, in the reverse direction (ATP hydrolysis), the chemical reaction drives the rotation of a c 10-14 subunit assembly followed by proton translocation. We discuss our very recent results regarding subunit rotation in Escherichia coli FoF1 with a combined biophysical and mutational approach.  相似文献   

6.
7.
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   

8.
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   

9.
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   

10.
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   

11.
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   

12.
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   

13.
A modified ‘cold chase’ technique was used to study tight [14C]ADP and [14C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF0F1). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF0F1 or CF1 reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k d of about 5 min−1. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF0F1. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF1 dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF1 becomes bound to a thylakoid membrane.  相似文献   

14.
Why Is the Mechanical Efficiency of F1-ATPase So High?   总被引:2,自引:0,他引:2  
The experimentally measured mechanical efficiency of the F1-ATPase under viscous loading is nearly 100%, far higher than any other hydrolysis-driven molecular motor (Yasuda et al., 1998). Here we give a molecular explanation for this remarkable property.  相似文献   

15.
V-type Na+-ATPase from Entercoccus hirae consists of nine kinds of subunits (NtpA3, B3, C1, D1, E1−3, F1−3, G1, I1, and K10) which are encoded by the ntp operon. The amino acid sequences of the major subunits, A, B, and K (proteolipid), were highly similar to those of A, B, and c subunits of eukaryotic V-ATPases, and those of β, α, and c subunits of F-ATPases. We modeled the A and B subunits by homology modeling using the structure of β and α subunits of F-ATPase, and obtained an atomic structure of NtpK ring by X-ray crystallography. Here we briefly summarize our current models of the whole structure and mechanism of the E. hirae V-ATPase.  相似文献   

16.
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   

17.
In Escherichia coli, the F1FO ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b arg36→ile and the b arg36→glu mutations. The b arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b arg36→glu mutation was largely suppressed in the b arg36→glu,glu39→arg double mutant. E. coli expressing the b arg36→glu,glu39→arg subunit grew well on succinate-based medium. F1FO ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F1FO ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.  相似文献   

18.
We have developed a novel micro-mixer using a biological molecular ATP motor. The micro-mixer was constructed from arrays of chromatophore-embedded δ-free F0F1-ATPases, where the δ-free F1 part acted as a rotator to mix solutions, and the F0 part was driven by light. Confocal microscope studies indicated that the micro-mixer did not touch directly on the fibrin labeled with FITC. The nanomechanical force generated by the motor induced drug movement in the solution and accelerated the fibrinolysis process. All results strongly suggest that the micro-mixers generated a nanomechanical force which accelerated the fibrinolysis process in the presence of lower concentrations of lumbrokinase.  相似文献   

19.
Summary Numerous Escherichia coli mutants have been used to determine the genetics and sequence of assembly of the prokaryotic proton-translocating ATP synthase complex. Similar studies with the analogous chloroplast ATP synthase in higher plants have not been possible due to lack of suitable mutants. We describe here a preliminary characterization of cfr, a nuclear mutation in Zea mays L. that appears to destabilize or prevent assembly of the chloroplast ATP synthase complex. Biochemical and physiological analyses indicate that the amounts of both the CF1 and CFo components of the complex are severely diminished. Mutant seedlings are pale green and occasionally survive, with greatly reduced vigor, to maturity. The cfr locus has been mapped genetically to the short arm of chromosome. 1.  相似文献   

20.
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   

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