17 Proton motive force

Dr. Padma Ambalam

PROTON MOTIVE FORCE

Objectives

To understand the function of Proton Motive Force in production of ATP.
To examine the importance of a proton gradients power the synthesis of ATP.
To examine the importance of shuttles for aerobic oxidation of cytosolic NADH.
To understand the cellular respiration which is regulated by the Need for ATP.

Introduction

A proton-motive force is “energy that is generated by the transfer of protons or electrons across an energy-transducing membrane and can be used for chemical, osmotic or mechanical work.”

It can be “generated by a variety of phenomena including the operation of an electron transport chain, illumination of a purple membrane, and the hydrolysis of ATP by a proton ATPase.”

Proton Gradient

Figure-1. Proton motif force

( Adapted from- http://classroom.sdmesa.edu/eschmid/Lectur46.gif)

Why Proton Gradients Are Necessary

The reason proton gradients are required boils back down to chemistry. In other words, to convert carbon dioxide into organic molecules, life attaches hydrogen atoms to CO2

This pathway is exothermic (releasing energy that can be captured as ATP) right through to pyruvate, one of the central molecules in cell metabolism

But there’s a problem, pointed out by William Martin in collaboration with Russell. All cells that use the acetyl-CoA pathway today depend on proton gradients. None of them can grow by fermentation

Why not? Because CO2 is a stable molecule and does not react easily, even with hydrogen – even when thermodynamics says it should react. CO2 is a bit like oxygen in this respect: Once it starts to react, it’s not easily stopped. But a fire needs a spark to get it going, and so, too, does CO2. Cells need the equivalent of a spark to get CO2 to react, and for cells, that spark is ATP

The problem is that the reaction of CO2 with H2 releases energy, but not a lot — only enough to make 1 ATP. That means cells have to spend 1 ATP to gain 1 ATP, so there’s no net gain. If there’s no gain, there’s no growth; no growth, no life

Gradients break that cycle. It’s not quite true to say that the reaction of CO 2 with H2 releases enough energy to make 1 ATP: it’s actually enough to make 1.5 ATPs. But of course there’s no such thing as 1.5 ATPs, at least not by stoichiometric chemistry; so the spare energy from the reaction, as chemistry, is lost

But that doesn’t happen with a gradient. In principle, a reaction can be repeated over and over again, just to pump a proton over a membrane

When enough protons have accumulated, the proton motif force powers the formation of ATP

So a gradient allows cells to save up protons as “loose change”, and that makes all the difference in the world — the difference between growth and no growth, life and no life.

A proton gradient powers the synthesis of ATP

The overall process of ATP generation via the harnessing of a proton motif force is called chemiosmosis
The Chemiosmotic Theory Proposed by Peter Mitchell in the 1960’s
The proton gradient generated by the oxidation of NADH and FADH2 is called the proton-motif force.The proton-motif force powers the synthesis of ATP

Coupled nature of respiration in mitochondria

(a) O2 consumed only with ADP, excess Pi.

(b) (+) Uncoupler DNP (O2 consumed without ADP).

(A) O2 consumed only with ADP, excess Pi

Oxidation of substrates is coupled to the phosphorylation of ADP
Respiration (consumption of oxygen) proceeds only when ADP is present
The amount of O 2 consumed depends upon the amount of ADP added

(B) (+) Uncoupler DNP (O 2 consumed without ADP)

Uncouplers stimulate the oxidation of substrates in the absence of ADP.
Uncouplers are lipid-soluble weak acids.
Both acidic and basic forms can cross the inner mitochondrial membrane.
Uncouplers deplete any proton gradient by transporting protons across the membrane.

2,4-Dinitrophenol: an uncoupler

Mitchell’s postulates for Chemiosmotic theory

Intact inner mitochondrial membrane is required (to maintain a proton gradient)
Electron transport through the ETC generates a proton gradient (pumps H + from the matrix to the intermembrane space)
The membrane-spanning enzyme, ATP synthase, catalyzes the phosphorylation of ADP in a reaction driven by movement of H + across the inner membrane into the matrix

Shuttles are Necessary for Aerobic Oxidation of Cytosolic NADH

Cytosolic NADH must enter the mitochondria to fuel oxidative phosphorylation, but NADH and NAD+ cannot diffuse across the inner mitochondrial membrane
Two shuttle systems for reducing equivalents:

Glycerol phosphate shuttle: insect flight muscles
Malate-aspartate shuttle: predominant in liver and other mammalian tissues

1. Glycerol phosphate shuttle

The glycerol-3-phosphate shuttle is a mechanism that regenerates NAD+ from NADH, a by-product of glycolysis. Its importance in transporting reducing equivalents is secondary to the malate-aspartate shuttle

Mechanism:

In this shuttle, the enzyme glycerol-3-phosphate dehydrogenase 1 (GPDH-C) converts dihydroxyacetone phosphate (2) to glycerol 3-phosphate (1) by oxidizing one molecule of NADH to NAD+

Glycerol-3-phosphate gets converted back to dihydroxyacetone phosphate by an inner membrane-bound mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPDH-M), this time reducing one molecule of enzyme-bound flavin adenine dinucleotide (FAD) to FADH2. FADH2 then reduces coenzyme Q (ubiquinone to ubiquinol) which enters into oxidative phosphorylation. This reaction is irreversible.

The glycerol-3-phosphate shuttle allows the NADH synthesized in the cytosol by glycolysis to contribute to the oxidative phosphorylation pathway in the mitochondria to generate ATP. It has been found in animals, fungi, and plants.

Figure -2 Glycerol phosphate shuttle

(Adapted from: http://oregonstate.edu/instruct/bb451/winter14/stryer7/CH18/figure_18_34.jpg))

Malate-aspartate shuttle

The malate-aspartate shuttle is a biochemical system for translocating electrons produced during glycolysis across the semipermeable inner membrane of the mitochondrion for oxidative phosphorylation in eukaryotes. These electrons enter in the electron transport chain of the mitochondria via reduction equivalents to generate ATP.

Mechanism:

The primary enzyme in the malate-aspartate shuttle is malate dehydrogenase.

First, in the cytosol, malate dehydrogenase catalyses the reaction of oxaloacetate and NADH to produce malate and NAD+.

Once malate is formed, the first antiporter (malate-alpha-ketoglutarate)imports the malate from the cytosol into the mitochondrial matrix and also exports alpha-ketoglutarate from the matrix into the cytosol simultaneously.

Oxaloacetate is then transformed into aspartate by mitochondrial aspartate aminotransferase. Since aspartate is an amino acid, an amino radical needs to be added to the oxaloacetate.

The second antiporter (the glutamate-aspartate antiporter) imports glutamate from the cytosol into the matrix and exports aspartate from the matrix to the cytosol.

The net effect of the malate-aspartate shuttle is purely redox: NADH in the cytosol is oxidized to NAD+, and NAD+ in the matrix is reduced to NADH.

Since the malate-aspartate shuttle regenerates NADH inside the mitochondrial matrix, it is capable of maximizing the number of ATPs produced in glycolysis (3/NADH), ultimately resulting in a net gain of 38 ATP molecules per molecule of glucose metabolized.

Figure- 3 Malate-aspartate shuttle

( Adapted from- http://oregonstate.edu/instruct/bb451/winter14/stryer7/CH18/figure_18_35.jpg)

Regulation of Proton motif force

Protonmotif force ( D p) is the energy of the proton concentration gradient

Figure – 4. Proton concentration gradient in chemical and electrical condition.

(Adapted from – http://www.ccrc.uga.edu/~rcarlson/bcmb3100/Chap21)

ATP synthase

There are 3 active sites, one in each b subunit
The c-e-g unit forms a “rotor”
Rotation of the g subunit inside the a3b3 hexamer causes domain movements in the b-subunits, opening and closing the active sites
The a-b-g-a3b3 unit is the “stator” (the Fo channel is attached to a3b3 by the ab-d arm)

Figure – 5. ATP synthase.

(Adapted from https://s3.amazonaws.com/classconnection/281/flashcards/2541281/png/session_15_ _oxidative_phosphorylation_-_google_slides_(1)-14C6815A20153228B19.png)

F0F1 ATP Synthase uses the proton gradient energy for the synthesis of ATP
An F-type ATPase which generates ATP
Composed of a “knob-and-stalk” structure
F1 (knob) contains the catalytic subunits
F0 (stalk) has a proton channel which spans the membrane.
Passage of protons through the F0 (stalk) into the matrix is coupled to ATP formation
Estimated passage of 3 H+ / ATP synthesized

Passage of protons through the F0 channel causes the rotor to spin in one direction and the stator to spin in the opposite direction

Three protons = one complete rotation = one ATP

Figure – 6 Conformational changes in ATP synthase.

1. ADP, Pi bind to an open site.

2. Inward passage of protons, conformation change, ATP synthesis from ADP and Pi.

3. ATP released from open site, ADP and Pi form ATP in the tight site.

(Adapted from http://1.bp.blogspot.com/_DZH2cmCoois/R2hL3wt_O6I/AAAAAAAAEHU/7WNKoQjakDk/s400/tmp.jpg)

Proton flow around the “c” ring

The number of c rings determines the number of protons required to synthesize a molecule of ATP.
The c ring of vertebrates consist of 8 subunits, making vertebrate ATP synthase the most efficient known.

Figure – 7 Proton flow around the C ring.

(Adapted from- https://s3.amazonaws.com/classconnection/281/flashcards/2541281/png/session_15_- _oxidative_phosphorylation_-_google_slides_(5)-14C68FE9F127B4A4690.png)

SUMMARY

It is generally accepted that the chemistry of water was the most crucial determinant in shaping life on earth. Among the more important chemical features of water is its dissociation into protons and hydroxyl ions.

The presence of relatively high proton concentrations in the ambient solution resulted in the evolution of proton pumps during the dawn of life on earth. These proton pumps maintained neutral pH inside the cells and generated electrochemical gradients of protons (proton-motive force) across their membranes.

The existence of proton-motif force enabled the evolution of porters driven by it that are most probably among the more primitive porters in the world. The directionality of the substrate transport by the porters could be to both sides of the membranes because they can serve as proton symporters or antiporters.

One of the most important subjects of this meeting is the mechanism by which proton-motif and other ion-motif forces drive the transport processes through porters.

Proton-motif force: free energy change during movement across a proton gradient

“Proton-motif force” (Dp) is a Dy- or DE-like term (DE is electromotive force ) that combines the concentration and voltage effects of a proton gradient such that

DG = – nFDp0′ + nFDp, and Dp0′ = 0 (proton motive force = 0 under std conds), so

DG = nF Dp

DG can also be expressed as the sum of the DpH and Dym contributions:

DG = – 2.303 RT DpH + nFDym

so nFDp = – 2.303 RT DpH + nFDym

Dp = (-2.303 RT DpH)/nF + Dym

This way of expressing the proton-motif force was probably adopted because of its elegant resemblance to the Nernst equation, but the clearest expression of the energy available from a proton gradient is probably DG = – 2.303 RT DpH + nFDym

Remember (this is a recording) that DpH = pHin – pHout and Dym = yin – yout because we started out by considering the movement of protons from the cytoplasm to the matrix:

The total free energy available from the movement of 1 mole of protons from the cytoplasm to the matrix under cellular conditions (DpH = 1.4, Dy = 0.14 V) is the sum of the free energy changes calculated in sections B and C:

Figure – 8. Transfer of Proton from the cytoplasm to mitochondria

( Adapted from- http://www.ccrc.uga.edu/~rcarlson/bcmb3100/Chap21)

Figure- 9 Many Mitochondrial Transporters

(Adapted from-http://www.ccrc.uga.edu/~rcarlson/bcmb3100/Chap21)

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References

Web site

Books

Harper’sIllustrated Biochemistry by Robert K.. Murray, Daryl K. Granner, Peter A. Mayes, 26th Edition (2003)
Lehninger’S Principle of Biochemistry David L. Nelson and Michael M. Cox,5th Edition (2008)
Instant Notes: Biochemistry, 2nd Edition, B.D. Hames & N. M. Hooper (2005)