Related Experiment Video
Updated: Aug 10, 2026
10:39
Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography
Published on: September 14, 2014
Rotation of the epsilon subunit during catalysis by Escherichia coli FOF1-ATP synthase
V V Bulygin1, T M Duncan, R L Cross
1Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210, USA.
The Journal of Biological Chemistry
|November 21, 1998
Summary
The epsilon subunit of FOF1-ATP synthase rotates during ATP synthesis and hydrolysis. This rotation is essential for coupling proton transport to energy production.
Area of Science:
- Biochemistry
- Molecular Biology
- Bioenergetics
Background:
- FOF1-ATP synthase is a crucial enzyme complex responsible for cellular energy production.
- The precise mechanism of energy transduction, particularly the role of individual subunits, remains an active area of research.
Purpose of the Study:
- To investigate the rotational dynamics of the epsilon subunit within the FOF1-ATP synthase during catalysis.
- To determine if the epsilon subunit functions as part of the enzyme's rotational mechanism.
Main Methods:
- Site-directed mutagenesis to introduce cysteine residues at specific positions (beta380 and epsilon108) for cross-linking.
- Disulfide bond formation to create specific cross-links between beta and epsilon subunits.
- Subunit dissociation/reassociation procedures to create hybrid F1 complexes with epitope-tagged subunits.
- Functional assays measuring ATP synthesis and hydrolysis.
- Reconstitution of hybrid F1 complexes with FO in membranes.
- Inhibition studies using N,N'-dicyclohexylcarbodiimide (DCCD).
Main Results:
- Specific beta-epsilon cross-linking was achieved, and tethering epsilon to beta subunits inhibited enzyme activity.
- Restoration of enzyme activity was observed upon reduction of the beta-epsilon cross-link.
- Experiments with epitope-tagged subunits demonstrated that epsilon preferentially cross-linked with a specific beta subunit after catalytic turnover, indicating rotation.
- Ligand binding without catalysis did not result in this specific cross-linking.
- Inhibition of proton translocation through FO abolished catalysis-dependent epsilon rotation.
Conclusions:
- The epsilon subunit undergoes rotation relative to the catalytic beta subunits during FOF1-ATP synthase function.
- The epsilon subunit is an integral component of the rotor that couples proton translocation to ATP synthesis/hydrolysis.
- These findings provide direct evidence for the rotational mechanism of ATP synthase.
Related Concept Videos
01:58Mechanical Protein Functions
Proteins perform many mechanical functions in a cell. These proteins can be classified into two general categories- proteins that generate mechanical forces and proteins that are subjected to mechanical forces. Proteins providing mechanical support to the structure of the cell, such as keratin, are subjected to mechanical force, whereas proteins involved in cell movement and transport of molecules across cell membranes, such as an ion pump, are examples of generating mechanical force.
01:48ATP Synthase: Mechanism
In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased ATP...
01:18ATP Synthase: Structure
ATP synthase or ATPase is among the most conserved proteins found in bacteria, mammals, and plants. This enzyme can catalyze a forward reaction in response to the electrochemical gradient, producing ATP from ADP and inorganic phosphate. ATP synthase can also work in a reverse direction by hydrolyzing ATP and generating an electrochemical gradient. Different forms of ATP synthases have evolved special features to meet the specific demands of the cell. Based on their specific feature, ATP...
01:34ATP Driven Pumps II: P-type Pumps
The P-type pumps are a large family of integral membrane transporter ATPases. They are divided into five major types based on substrate specificity, from I to V.
A typical P-type pump has three cytosolic domains: nucleotide-binding (N), phosphorylation (P), and activator (A) domains. These domains are connected to the membrane-spanning helices by short amino acid segments. ATP hydrolysis and covalent phosphoenzyme intermediate formation are crucial parts of the catalytic cycle. At the highly...
A typical P-type pump has three cytosolic domains: nucleotide-binding (N), phosphorylation (P), and activator (A) domains. These domains are connected to the membrane-spanning helices by short amino acid segments. ATP hydrolysis and covalent phosphoenzyme intermediate formation are crucial parts of the catalytic cycle. At the highly...
01:29Electron Transport Chain Components
The electron transport chain (ETC) is a crucial metabolic pathway that facilitates energy conversion in prokaryotic and eukaryotic cells. In eukaryotes, the ETC comprises four membrane-associated protein complexes in the inner mitochondrial membrane. In prokaryotes, the ETC in the plasma membrane can vary in composition, with fewer or different complexes depending on the organism and environmental conditions. These complexes transfer electrons from electron donors, such as NADH and FADH2, to...
01:22Chemiosmosis and ATP Synthesis
The electron transport chain is a critical component of cellular respiration, occurring in the inner mitochondrial membrane. It facilitates the transfer of high-energy electrons from reduced cofactors NADH and FADH₂ to molecular oxygen, the final electron acceptor. This transfer of electrons through a series of protein complexes is tightly coupled to the translocation of protons across the membrane, generating a proton gradient essential for ATP synthesis.Electron Flow and Proton...