Assembled F1-(αβ) and Hybrid F1-α3β3γ-ATPases fromRhodospirillum rubrum α, Wild Type or Mutant β, and Chloroplast γ Subunits

Du, Ziyun; Tucker, Ward C.; Richter, Mark L.; Gromet-Elhanan, Zippora

doi:10.1074/jbc.m007568200

Cited by 20 publications

(20 citation statements)

References 47 publications

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“…This finding is on par with earlier reports that the MgATPase activity of both CF 1 (16) and RF 1 (17) is very sensitive to inhibition by free Mg 2 , and in both cases high rates of catalysis require addition of oxyanions such as bicarbonate or sulfite (19,31). In addition, at 22°C under the conditions of the rotation assay, the MgATPase activity of the ␣ R 3 ␤ R 3 ␥ C complex was 4-fold lower than its CaATPase activity (Table I).…”

Section: Resultscontrasting

confidence: 49%

“…The complex fully retained the unique properties of its parent ATPases, which include sensitivity to inhibition by free magnesium (16,17), and a higher CaATPase than MgATPase activity (Table I), both being modulated by ␥ C oxidation and reduction (19,27). Single-molecule studies of extracted native CF 1 (lacking a ␦ subunit) have already provided some evidence for its ␥ subunit rotation (23).…”

Section: Resultsmentioning

confidence: 99%

“…We have recently assembled in our laboratory a hybrid photosynthetic F 1 -ATPase consisting of RF 1 -␣ R and ␤ R subunits and a CF 1 -␥ C subunit, all expressed in E. coli (19). The isolated hybrid ␣…”

Section: Resultsmentioning

confidence: 99%

“…C complex, and purified by high performance liquid chromatography as described by Du et al (19). The purity of the isolated hybrid complex was evaluated by SDS-PAGE (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Understanding the mechanism by which calcium decouples ATP hydrolysis and proton translocation can enhance our knowledge of the interaction between various parts of the F 0 F 1 molecule in relation to its functionality. We provide here the first step toward this goal by demonstrating that the assembled hybrid photosynthetic F 1 in our laboratory (19), shows rotational motion in the presence of CaATP. This result indicates that the decoupling effect of calcium, which does not interrupt the connection between ATP hydrolysis and ␥ C rotation, might be mediated by a long range effect on the membrane bound F 0 subunits.…”

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confidence: 99%

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Observation of Calcium-dependent Unidirectional Rotational Motion in Recombinant Photosynthetic F1-ATPase Molecules

Tucker

Schwarz

Levine

et al. 2004

Journal of Biological Chemistry

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ATP hydrolysis and synthesis by the F 0 F 1 -ATP synthase are coupled to proton translocation across the membrane in the presence of magnesium. Calcium is known, however, to disrupt this coupling in the photosynthetic enzyme in a unique way: it does not support ATP synthesis, and CaATP hydrolysis is decoupled from any proton translocation, but the membrane does not become leaky to protons. Understanding the molecular basis of these calcium-dependent effects can shed light on the as yet unclear mechanism of coupling between proton transport and rotational catalysis. We show here, using an actin filament ␥-rotation assay, that CaATP is capable of sustaining rotational motion in a highly active hybrid photosynthetic F 1 -ATPase consisting of ␣ and ␤ subunits from Rhodospirillum rubrum and ␥ subunit from spinach chloroplasts (␣ Recent experiments have provided strong evidence for the rotational catalysis mechanism proposed for the ubiquitous enzyme F 0 F 1 -ATPase/ATP synthase (1, 2). Particularly striking is the effort led by several laboratories to measure the rotational motion of the F 1 part of the enzyme on the singlemolecule level. F 1 is constructed of five subunits, with a stoichiometry of ␣ 3 ␤ 3 ␥␦⑀. Fluorescent actin filaments attached to the ␥ subunit of engineered surface-immobilized ␣ 3 ␤ 3 ␥ subcomplexes of the enzyme, either from a thermophyilic bacterium, TF 1 1 (3), or from Escherichia coli, EF 1 (4, 5), were shown to rotate unidirectionally while hydrolyzing MgATP. This rotation was not only dependent on ATP concentration but was shown to have almost 100% efficiency of chemical to mechanical energy conversion. At very low ATP concentration the stepwise motion of the enzyme was exposed (6). Small beads attached to ␥ in place of the actin filament allowed Yasuda et al.(7) to follow the motion at the limit of small load and watch rotation at a rate as high as 130 revolutions per second. Most spectacularly, forced clockwise rotation was shown very recently to lead to ATP synthesis, thus confirming the essential role of ␥ subunit rotation in both directions of the reaction (8). Some single-molecule work also addressed the operation of the F 0 part of the F 0 F 1 -ATP synthase. It was shown that the F 0 -c subunit oligomer, which consists of 10 -14 identical c subunits, is capable of rotating together with ␥ (9, 10).While much new information has been collected regarding rotational catalysis in the ATP synthase, there are still many open questions, mainly in relation to the coupling between enzymatic activity and rotational motion. The photosynthetic version of the ATP synthase is an attractive system to probe some of these questions, since it presents several distinct functional properties. These include the tight regulation of ATP hydrolysis, which is especially important in photosynthetic cells, where it prevents the depletion of essential ATP pools in the dark (11-13). Indeed, plant chloroplasts have a unique regulatory system, termed thiol modulation, which leads to reduction of a disulfide bond fo...

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Section: Resultscontrasting

confidence: 49%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…C complex, and purified by high performance liquid chromatography as described by Du et al (19). The purity of the isolated hybrid complex was evaluated by SDS-PAGE (Fig.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Observation of Calcium-dependent Unidirectional Rotational Motion in Recombinant Photosynthetic F1-ATPase Molecules

Tucker

Schwarz

Levine

et al. 2004

Journal of Biological Chemistry

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F‐ATP synthase and the permeability transition pore: fewer doubts, more certainties

et al. 2019

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Whether the mitochondrial permeability transition pore (PTP), also called mitochondrial megachannel (MMC), originates from the F‐ATP synthase is a matter of controversy. This hypothesis is supported both by site‐directed mutagenesis of specific residues of F‐ATP synthase affecting regulation of the PTP/MMC and by deletion of specific subunits causing dramatic changes in channel conductance. In contrast, human cells lacking an assembled F‐ATP synthase apparently display persistence of the PTP. We discuss recent data that shed new light on this controversy, supporting the conclusion that the PTP/MMC originates from a Ca2+‐dependent conformational change in F‐ATP synthase allowing its reversible transformation into a high‐conductance channel.

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Ca²⁺ as cofactor of the mitochondrial H⁺‐translocating F₁F_O‐ATP(hydrol)ase

Nesci

Pagliarani

2021

Proteins

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The mitochondrial F1FO‐ATPase in the presence of the natural cofactor Mg2+ acts as the enzyme of life by synthesizing ATP, but it can also hydrolyze ATP to pump H+. Interestingly, Mg2+ can be replaced by Ca2+, but only to sustain ATP hydrolysis and not ATP synthesis. When Ca2+ inserts in F1, the torque generation built by the chemomechanical coupling between F1 and the rotating central stalk was reported as unable to drive the transmembrane H+ flux within FO. However, the failed H+ translocation is not consistent with the oligomycin‐sensitivity of the Ca2+‐dependent F1FO‐ATP(hydrol)ase. New enzyme roles in mitochondrial energy transduction are suggested by recent advances. Accordingly, the structural F1FO‐ATPase distortion driven by ATP hydrolysis sustained by Ca2+ is consistent with the permeability transition pore signal propagation pathway. The Ca2+‐activated F1FO‐ATPase, by forming the pore, may contribute to dissipate the transmembrane H+ gradient created by the same enzyme complex.

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Assembled F1-(αβ) and Hybrid F1-α3β3γ-ATPases fromRhodospirillum rubrum α, Wild Type or Mutant β, and Chloroplast γ Subunits

Cited by 20 publications

References 47 publications

Observation of Calcium-dependent Unidirectional Rotational Motion in Recombinant Photosynthetic F1-ATPase Molecules

Observation of Calcium-dependent Unidirectional Rotational Motion in Recombinant Photosynthetic F1-ATPase Molecules

F‐ATP synthase and the permeability transition pore: fewer doubts, more certainties

Ca²⁺ as cofactor of the mitochondrial H⁺‐translocating F₁F_O‐ATP(hydrol)ase

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Assembled F1-(αβ) and Hybrid F1-α3β3γ-ATPases fromRhodospirillum rubrum α, Wild Type or Mutant β, and Chloroplast γ Subunits

Cited by 20 publications

References 47 publications

Observation of Calcium-dependent Unidirectional Rotational Motion in Recombinant Photosynthetic F1-ATPase Molecules

Observation of Calcium-dependent Unidirectional Rotational Motion in Recombinant Photosynthetic F1-ATPase Molecules

F‐ATP synthase and the permeability transition pore: fewer doubts, more certainties

Ca2+ as cofactor of the mitochondrial H+‐translocating F1FO‐ATP(hydrol)ase

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Ca²⁺ as cofactor of the mitochondrial H⁺‐translocating F₁F_O‐ATP(hydrol)ase