Biochemistry Online: An Approach Based on Chemical Logic

Biochemistry Online





Learning Goals/Objectives for Chapter 8C: 

After class and this reading, students will be able to

  • explain reasons for the strongly exergonic hydrolysis of carboxylic acid anhydrides, phosphoric acid anhydrides, mixed anhydrides, and analogous structures and give approximate  values for the ΔG0 of hydrolysis of them;
  • identify from Lewis structures molecules whose hydrolytic cleavage are strongly exergonic;
  • explain how the exergonic cleavage of phophoanhydride bonds in ATP can be coupled to the endergonic synthesis of macromolecules like proteins;
  • draw mechanisms to show how oxidation and phosphorylation reactions are coupled in anaerobic metabolism through the productions of a mixed anhydride catalyzed by the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase;
  • explain how arsenate can double oxidation and phosphorlyation reactions in glycolysis
  • explain how NAD+ can be regenerated from NADH in anaeroboic condition to allow glycolysis to continue;
  • explain the general flow of electrons from NADH to dioxgen through a series of mobile and membrane protein bound electron acceptors in electron transport in the mitochondria inner member.
  • explain with picture diagrams how oxidation and phosphorylation reactions (to produce ATP) are coupled in aerobic metabolism through the generation and collapse of a proton gradient in the mitochondria;
  • draw pictures diagrams explaining the structure of F1F0ATPase in the inner mitochondria member and explain using the picture how ATP synthesis is coupled to protein gradient collapse
  • write an equation for the electrochemical potential and use it to calculate the available ΔG0 for ATP production on proton gradient collapse, given typical values for ΔpH and ΔE across the membrane 

C10.  Proton Gradient Collapse and ATP synthesis - Structure

The mechanism by which the proton gradient drives ATP synthesis involves a complex coupling of the F0 and F1 subunits.  A more detailed image of the whole ATO synthase complex is shown below.

Figure:  Detailed View of F0F1 ATP synthase structure

F0F1 Detailed Structure

Closer views of the c subunits and a yellow rectangle representing the a subunit (missing in the combined crystal structure) comprise the Fo part of the complex are shown below.  These subunits reside in the inner membrane of the mitochondria (or cell membrane of bacteria) and are involved in proton transport from matrix (or cytoplasm of a bacteria) to the inner membrane space (or periplasmic space of bacteria).  The multiple c subunits consist of two very hydrophobic helices connected by a loop in a helix-loop-helix motif. 

c subunit ATP Synthase Proton flow

New 7/127/17:  The following Jmol links contains multiple views of the FoF1ATPase.  It is repeated several times below.

Jmol:   Bovine F0F1 ATPase aka ATP Synthase (under development)  Jmol14 (Java) |  JSMol  (HTML5)



Two classic inhibitors (structures shown below) of ATP synthase interact with the Fo subunit.  One, oligomycin A, binds between the a and c subunits and blocks proton transport activity of the Fo subunit. Oligomycin A sensitivity requires, paradoxically, OSCP (Oligomycin-Sensitivity Conferring Protein which is analogous to the bacterial delta subunit), a stalk protein subunit distal to Fo which couples Fo and F1.   Another inhibitor, dicyclohexylcarbodiimide reacts with a protonated Asp 61 in c subunits of F0.  It does so even at pH 8.0 which indicates that the pKa of the Asp 61 is much higher than usual. This might occur if the Asp is a very hydrophobic environment. The modification of one As 61 in only one c subunit is necessary to stop Fo activity.   The protonated carboxyl group donates a proton to a N atom in DCCD, which then reacts with the deprotonated Asp to form an O-acyl isourea derivative.

Figure: Structure of Oligomycin A and DCCD - Inhibitors of proton transport by Fo

Oligomycin A and DCCD


The figures below shown the structure of the ac complex from E. Coli.  Protons flow to the a chain Arg 210 which is between two Asp 61 on adjacent c chains.  One of the Asp 61 is protonated allowing it to alter conformation and essentially rachet in the membrane domain in a motional faciliated by the development of a neutral protonated Asp.   


Protons from the inner membrane space or in the periplasmic space (in the above figures) then flow from the periplasm by forming a "handshaking" proton transfer relay which delivers another proton to the deprotonated Arg 210 allowing the circular ratching of the c subunits in the membrane to continue.  A set of polar residues entirely within subunit a, including Gln 252, Asn 214, Asn 148, Asp 119, His 245, Glu 219, Ser 144 and Asn 238 provide the path as illustrated below. 

H path ATP synthase E Coli

 When a proton is passed to the unprotonated Asp  61, a conformational change in the protonated c subunit occurs.  This leads to changes in c subunit interactions which seems to ratchet the c12 core.  Since the c12 oligomer  contacts  the γsubunit connecting the Fo stalk and F1 ATPase units, the γ subunit rotates, leading to sequential conformational changes in each of the 3 contacted  (αβ)2 dimers of the F1 enzyme. This leads to changes in ATP affinity through cycling each through the L, O, and T conformations. 

Figure:  Coupling Proton Flow in F0 to Conformation Change


Reprinted by permission of Nature. Rastogi & Girvin.  Nature 402, 263-268 (1999) Copyright 1999 McMilllan Publishers LTD

animation 1: ATP synthesis

animation 2: ATP synthesis mechanism

Molecular Movies (scroll down to ATP synthase)

Mitochondria:  Harvard

ATP sythase

Powering the Cell: Mitochondria

XVIVO: Scientific Animations

ATPase animations from the MRC

General List of Movies

In summary, FOF1ATPase (or synthase) is a rotary enzyme that ultimately couples collapse of a proton gradient (a chemical potential gradient which contributes to the transmembrane electrical potential) to a chemical (phosphorylation) step.  The rotor, which is in contact with both the FO  proton pore, and the F1 synthase, moves with respect to both subunits, which couples them.  Motion of course is relative so the rotor can be thought of as static with the FO and F1 subunits as rotating.   The FO pore can hence to be considered an electrical motor and the F1 synthase a chemical motor.  Carrying the analogy of a motor even further, the FO electrical motor turns the F1 chemical motor into a generator, not of electricity but of ATP.  The figure and link below, taken from the Protein Data Bank, go into more depth about this nanomotor.

Figure: ATP synthase Complex

ATP synthase from the PDB


Return to Chapter 8C:  ATP and Oxidative Phosphorylation

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Archived version of full Chapter 8C:  ATP and Oxidative Phosphorylation


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