C10. Proton Gradient Collapse and ATP synthesis - Structure - Biology

C10.  Proton Gradient Collapse and ATP synthesis - Structure - Biology

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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

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.

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

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.

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

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.

  • ATP synthase from the PDB

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Intracellular pH regulation controls energy balance and cell proliferation: chemical and biological proof of principle

Chemical proof of principle

Biological proof of principle: the role of the Na + /H + exchanger-1

Oncogene activation and transformation cause acidosis

Warburg effect (aerobic glycolysis)

Inhibition of tumour suppressor genes and oncogene activation drive the ‘Warburg effect’ and cause acidosis

Neoplastic transformation drives intracellular alkalinization and extracellular acidification through the activation and up-regulation of pHi-regulating systems

Hypoxia promotes acidosis by shifting from oxidative phosphorylation to glycolytic metabolism

HIF mediates cellular adaptation to low oxygen availability

HIF-induced metabolic reprogramming in response to tumour hypoxia causes acidosis

Acidosis may affect HIF-α stabilization and on HIF-induced gene regulation

Hypoxia enhances the expression and activity of pHi-regulating systems to promote cell survival and invasion

Hypoxia increases NHE-1 expression and activity

The hypoxia-induced membrane-associated carbonic anhydrases are key enzymes involved in pH homeostasis, cell survival and migration in a hypoxic/acidic microenvironment

CAIX regulation and expression

CAXII regulation and expression

The activity and functions of CAIX and CAXII

The hypoxia-induced monocarboxylate transporter MCT4, the constitutively expressed MCT1 and their chaperone CD147 are key plasma-membrane proteins involved in pH regulation, energy balance, tumour progression and metastasis

MCT regulation, expression, structure and implication of their chaperone CD147

The MCT1, MCT4 and CD147 activity and functions

Strategies taking advantage of changes in the oxygen level, energy balance and pH homeostasis to target primary tumours and metastases

Decreasing the pHi of hypoxic cells of the primary tumour by inhibiting key pHi-regulating systems to collapse ATP production

Increasing pHo and the extracellular buffering capacity in targeting metastasis and reducing multidrug resistance

Maintenance of cellular pH homeostasis is fundamental to life. A number of key intracellular pH (pHi) regulating systems including the Na + /H + exchangers, the proton pump, the monocarboxylate transporters, the HCO3 − transporters and exchangers and the membrane-associated and cytosolic carbonic anhydrases cooperate in maintaining a pHi that is permissive for cell survival. A common feature of tumours is acidosis caused by hypoxia (low oxygen tension). In addition to oncogene activation and transformation, hypoxia is responsible for inducing acidosis through a shift in cellular metabolism that generates a high acid load in the tumour microenvironment. However, hypoxia and oncogene activation also allow cells to adapt to the potentially toxic effects of an excess in acidosis. Hypoxia does so by inducing the activity of a transcription factor the hypoxia-inducible factor (HIF), and particularly HIF-1, that in turn enhances the expression of a number of pHi-regulating systems that cope with acidosis. In this review, we will focus on the characterization and function of some of the hypoxia-inducible pH-regulating systems and their induction by hypoxic stress. It is essential to understand the fundamentals of pH regulation to meet the challenge consisting in targeting tumour metabolism and acidosis as an anti-tumour approach. We will summarize strategies that take advantage of intracellular and extracellular pH regulation to target the primary tumour and metastatic growth, and to turn around resistance to chemotherapy and radiotherapy.


Since pHi regulation controls many cellular functions involved in energy production, cell survival, proliferation and migration, a strategy that would inhibit some of the major pHi-regulating systems of highly glycolytic cells, such as transformed cells and hypoxic tumour cells, is now being intensely investigated. Validation of the role of HIF-1-induced pHi-regulating systems such as CAIX, CAXII and MCT4 in tumorigenesis and tumour development is in progress. Thus, the challenge now consists in evaluating the most specific and promising drugs that target these systems, for possible combination with chemotherapeutic compounds.

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