Fast ATP-dependent subunit rotation in reconstituted FoF1-ATP synthase trapped in solution

Resonance Energy ◽

Atp Synthesis ◽

Electrochemical Potential ◽

Ensemble Averaging ◽

Subunit Rotation ◽

ABSTRACTFoF1-ATP synthases are the ubiquitous membrane enzymes which catalyze ATP synthesis or ATP hydrolysis in reverse, respectively. Enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. By utilizing an Anti- Brownian electrokinetic trap (ABEL trap), single-molecule Förster resonance energy transfer (smFRET)-based subunit rotation monitoring was prolonged from milliseconds to seconds. The extended observation times for single proteoliposomes in solution allowed to observe fluctuating rotation rates of individual enzymes and to map the broad distributions of ATP-dependent catalytic rates in FoF1-ATP synthase. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores and uncouplers the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second, i.e. much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 fragments without coupling to the membrane-embedded Fo domain of the enzyme.

Subunit movement in individual H+-ATP synthases during ATP synthesis and hydrolysis revealed by fluorescence resonance energy transfer

Biochemical Society Transactions ◽

10.1042/bst0330878 ◽

2005 ◽

Vol 33 (4) ◽

pp. 878-882 ◽

Cited By ~ 10

Author(s):

M. Börsch ◽

P. Gräber

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Single Molecule ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Resonance Energy ◽

Atp Synthesis ◽

Fluorescence Resonance ◽

F-type H+-ATP synthases synthesize ATP from ADP and phosphate using the energy supplied by a transmembrane electrochemical potential difference of protons. Rotary subunit movements within the enzyme drive catalysis in either an ATP hydrolysis or an ATP synthesis direction respectively. To monitor these subunit movements and associated conformational changes in real time and with subnanometre resolution, a single-molecule FRET (fluorescence resonance energy transfer) approach has been developed using the double-labelled H+-ATP synthase from Escherichia coli. After reconstitution into a liposome, this enzyme was able to catalyse ATP synthesis when the membrane was energized.

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

Molecules ◽

10.3390/molecules24030504 ◽

2019 ◽

Vol 24 (3) ◽

pp. 504 ◽

Cited By ~ 6

Author(s):

Hendrik Sielaff ◽

Seiga Yanagisawa ◽

Wayne D. Frasch ◽

Wolfgang Junge ◽

Michael Börsch

Keyword(s):

Single Molecule ◽

Binding Sites ◽

Atp Hydrolysis ◽

Resonance Energy ◽

Fluorescence Probes ◽

Structural Asymmetry ◽

Rotationally Symmetric ◽

Flow Through ◽

F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)3δab2. Subunits c10 or (αβ)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.

Twisting and subunit rotation in single F O F 1 -ATP synthase

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2012.0024 ◽

2013 ◽

Vol 368 (1611) ◽

pp. 20120024 ◽

Cited By ~ 28

Author(s):

Hendrik Sielaff ◽

Michael Börsch

Keyword(s):

Energy Transfer ◽

Single Molecule ◽

Resonance Energy ◽

Step Size ◽

Storage Mechanism ◽

Cellular Processes ◽

Recent Developments ◽

Subunit Rotation ◽

F O F 1 -ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single F O F 1 -ATP synthases.

Subunit rotation and twisting in FoF1-ATP synthase by single-molecule three-color Förster resonance energy transfer

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/j.bbabio.2012.06.030 ◽

2012 ◽

Vol 1817 ◽

pp. S8

Author(s):

Michael Börsch

Keyword(s):

Energy Transfer ◽

Atp Synthase ◽

Single Molecule ◽

Resonance Energy ◽

Forster Resonance Energy Transfer ◽

Förster Resonance Energy Transfer ◽

Subunit Rotation ◽

Fof1 Atp Synthase ◽

Förster Resonance

Structural dynamics of P-type ATPase ion pumps

Biochemical Society Transactions ◽

10.1042/bst20190124 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1247-1257 ◽

Cited By ~ 17

Author(s):

Mateusz Dyla ◽

Sara Basse Hansen ◽

Poul Nissen ◽

Magnus Kjaergaard

Keyword(s):

Structural Dynamics ◽

Single Molecule ◽

New Technologies ◽

Atp Hydrolysis ◽

Resonance Energy ◽

Time Resolved ◽

Dynamics Simulations ◽

Types Of Information ◽

P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Structure of a bacterial ATP synthase

10.1101/463968 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hui Guo ◽

Toshiharu Suzuki ◽

John L. Rubinstein

Keyword(s):

Atp Synthase ◽

Biochemical Analysis ◽

Atp Hydrolysis ◽

Genetic Manipulation ◽

Proton Translocation ◽

Atp Synthesis ◽

Mitochondrial Complex ◽

Rotational States ◽

Membrane Region ◽

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

A PLANT BIOCHEMIST'S VIEW OF H+-ATPases AND ATP SYNTHASES.

Journal of Experimental Biology ◽

10.1242/jeb.172.1.431 ◽

1992 ◽

Vol 172 (1) ◽

pp. 431-441

Author(s):

RE Mccarty

Keyword(s):

Plasma Membrane ◽

Atp Synthase ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Proton Gradient ◽

Sequencing Analysis ◽

Isolation And Purification ◽

Membrane Atpases ◽

Vacuolar Atpases ◽

My twenty-five year fascination with membrane ATPases grew out of my experiences in the laboratories of André Jagendorf and Efraim Racker. André introduced me to photosynthetic phosphorylation and Ef, to whose memory this article is dedicated, convinced me that ATPases had much to do with ATP synthesis. Astounding progress has been made in the H+-ATPase field in just two decades. By the early 1970s, it was generally recognized that oxidative and photosynthetic ATP synthesis were catalyzed by membrane enzymes that could act as H+-ATPases and that the common intermediate between electron transport and phosphorylation is the electrochemical proton gradient. At that time, it had been shown that a cation-stimulated ATPase activity was associated with plasma membrane preparations from plant roots. The endomembrane or vacuolar ATPases were unknown. The application of improved biochemical methods for membrane isolation and purification, as well as membrane protein reconstitutions, led rapidly to the conclusion that there are three major classes of membrane H+-ATPases, P, V and F. P-ATPases, which will not be considered further in this article, are phosphorylated during their catalytic cycle and have a much simpler polypeptide composition than V- or F-ATPases. The plasma membrane H+-ATPase of plant, yeasts and fungal cells is one example of this class of enzymes (see Pedersen and Carafoli, 1987, for a comparison of plasma membrane ATPases). Biochemical and gene sequencing analysis have revealed that V- and F-ATPases resemble each other structurally, but are distinct in function and origin. The 'V' stands for vacuolar and the 'F' for F1Fo. F1 was the first factor isolated from bovine heart mitochondria shown to be required for oxidative phosphorylation. Fo was so named because it is a factor that conferred oligomycin sensitivity to soluble F1. Other F-ATPases are often named to indicate their sources. For example, chloroplast F1 is denoted CF1 (see Racker, 1965, for early work on F1). Recent successes in reconstitution of vacuolar ATPase have led to a V1Vo nomenclature for this enzyme as well. The term 'ATP synthase' is now in general use to describe F-ATPases. This term emphasizes the facts that although F-ATPases function to synthesize ATP, they do not catalyze, normally, ATP hydrolysis linked to proton flux. In contrast, V-ATPases are very unlikely to operate as ATP synthases. Thus, F-ATPases are proton gradient consumers, whereas V-ATPases generate proton gradients at the expense of hydrolysis. In this brief review, I will compare the structures of F- and V-ATPases. Also, I give some insight into the mechanisms that help prevent wasteful ATP hydrolysis by the chloroplast ATP synthase (CF1Fo).

Structure of a bacterial ATP synthase

eLife ◽

10.7554/elife.43128 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 46

Author(s):

Hui Guo ◽

Toshiharu Suzuki ◽

John L Rubinstein

Keyword(s):

Atp Synthase ◽

Biochemical Analysis ◽

Atp Hydrolysis ◽

Genetic Manipulation ◽

Proton Translocation ◽

Atp Synthesis ◽

Mitochondrial Complex ◽

Rotational States ◽

Membrane Region ◽

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the Bacillus PS3 ATP synthase in Eschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit ε shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

Deleting the IF 1 -like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis

Open Biology ◽

10.1098/rsob.170206 ◽

2018 ◽

Vol 8 (1) ◽

pp. 170206 ◽

Cited By ~ 9

Author(s):

Febin Varghese ◽

James N. Blaza ◽

Andrew J. Y. Jones ◽

Owen D. Jarman ◽

Judy Hirst

Keyword(s):

Atp Synthase ◽

Atp Hydrolysis ◽

Paracoccus Denitrificans ◽

Atp Synthesis ◽

Membrane Vesicles ◽

Inhibitor Protein ◽

Wild Type ◽

Efficient Energy ◽

Inverted Membrane Vesicles ◽

In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ζ subunit, which is structurally analogous to the mammalian inhibitor protein IF 1 . Here, we used homologous recombination to delete the ζ subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F 1 -ATPase subcomplex. ATP synthesis was not affected by loss of the ζ subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ζ subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.

pH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism

eLife ◽

10.7554/elife.70016 ◽

2021 ◽

Vol 10 ◽

Author(s):

Seiga Yanagisawa ◽

Wayne D Frasch

Keyword(s):

Atp Synthase ◽

Single Molecule ◽

Atp Hydrolysis ◽

Proton Translocation ◽

Atp Synthesis ◽

Proton Pumping ◽

Subunit A ◽

Pka Values ◽

C Subunit ◽

Ph Dependent

Most cellular ATP is made by rotary F1FO ATP synthases using proton translocation-generated clockwise torque on the FO c-ring rotor, while F1-ATP hydrolysis can force counterclockwise rotation and proton pumping. The FO torque-generating mechanism remains elusive even though the FO interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F1FO structures. Here, single-molecule F1FO rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of FO to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through FO operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the E. coli c10-ring of F1FO against the torque of F1-ATPase-dependent rotation that result from H+ transfer events from FO subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c10-ring occur to sustain F1FO ATP synthesis.