Metabolic Minimap

Mitochondrial ATP Formation

Received for publication, December 13, 2001

Donald Nicholson‡

From the School of Biochemistry and Molecular Biology, The University of Leeds,Leeds LS2 9JT, United Kingdom

This minimap is perhaps the second most vital of themall (the first, of course, being photosynthesis), but its fairlyobvious title has been the subject of much thought. Theobject has been to interrelate three pathways in whichenergy generated by respiration is coupled to the synthe-sis of ATP from ADP and phosphate. The formation of ATPis the end point, the consummation, of catabolism and isthe major purpose of what in our metabolic pathways chartwe designate and identify as the backbone of metabolism.This starts with glycolysis, which takes place in the cyto-plasm, and leads on to pyruvate dehydrogenase, the citricacid cycle, and oxidative phosphorylation, all of which takeplace in the mitochondria. Oxidative phosphorylation,however, is no longer an appropriate description of the twointerrelated but separated sequences that lead to ATPformation. The phosphorylation is not itself oxidative butdepends on the product of the preceding oxidative se-quence i.e. translocated protons. The (oxidative) electro-motive proton translocation provides the proton motiveforce that then drives ATP synthesis. Mitochondrial ATPformation seemed to be a more accurate description forthe whole process than ATP synthesis, because there are14 prior reactions in the synthesis of ADP. Three pathwaysare shown in this minimap (Fig. 1), the citric acid cycle,including pyruvate oxidation, aerobic oxidation of NADHand succinate, and ATP synthesis. Some relevant, andhopefully controversial, points about each are nowconsidered.

THE CITRIC ACID CYCLE, INCLUDING PYRUVATE OXIDATION

This starts with pyruvate derived mainly as the productof glycolysis of carbohydrates in the cytoplasm. This isoxidatively decarboxylated to acetyl-CoA, which is alsothe product of lipid catabolism within the mitochondrialmatrix. Acetyl-CoA enters the citric acid cycle where itundergoes four oxidation reactions resulting in the forma-tion of three NADH and one succinate, together with CO2

and water.The vital importance of NADH formation is accentuated

in the map by red arrows, and this led to a search for theorigin of its hydride ions, also intended to be shown in red.In the case of isocitrate and malate the answer seemedclear; straightforward dehydrogenation! However, in

malate both hydrogens come from water in the previousreaction. I never anticipated any problems with the pyru-vate and oxoglutarate dehydrogenase reactions. I hadbeen happy to live with the citric acid cycle for 60 years buthad never really thought about this part of it before. Wheredo the hydride ions in the NADH come from? There is nostructural hydrogen that can be utilized, and whereas co-enzymes may be a transient source they cannot (by defi-nition) be the ultimate. Although the electrons of the hy-dride ion come from the carbon skeletons of thesubstrates being oxidized, the protons must come fromwater. I consulted my text books, but none asked, let aloneanswered the question, so I consulted colleagues. Theythought about it and then (nearly, but not all) acquiesced,sometimes reluctantly, and some thought it of little impor-tance. However, most of the textbooks do discuss themore academic question of the origin of the CO2 evolved inthe citric acid cycle but ignore what seems to me to bemuch more important reactions involving the fundamentalpurpose of the cycle, the provision of NADH. The signifi-cance of water in biochemistry can often fail to beappreciated.

All the reactions of the citric acid cycle take place in themitochondrial matrix with the exception of succinic dehy-drogenase, which is part of Complex II of the inner mem-brane. It is important not to regard FADH2 as the productof this reaction, which is still often done. FAD is the first,but only a transient, carrier of electrons from succinate toubiquinone. Indeed the official name of the enzyme issuccinate dehydrogenase (ubiquinone).

AEROBIC OXIDATION

The mitochondrion is often regarded as the powerhouseof the cell, and this designation becomes much moremeaningful if we remember that a flow of electrons is anelectric current, and NADH and succinate provide the fuelfor an electricity generator. The pathway is often called theelectron transport chain, but its function is to create a flowof electrons (shown in Fig. 1 as heavy red arrows) toprovide the energy needed to translocate protons from themitochondrial matrix to the intermembrane space (shownin Fig. 1 as heavy blue arrows).

Four complexes are involved. Complexes I and II arelinked to Complex III via the Q-cycle and Complexes III orIV via cytochrome c. Proton translocation takes place inComplexes I, III, and IV (approximately) as shown.

‡ To whom correspondence should be addressed. E-mail:donald.nicholson@bmb.leeds.ac.uk.

© 2002 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATIONPrinted in U.S.A. Vol. 30, No. 1, pp. 3–5, 2002

This paper is available on line at http://www.bambed.org 3

FIG. 1. A metabolic minimap of mitochondrial ATP formation.

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ATP SYNTHASE

This is the all-important reaction in which the protonmotive force produced by proton translocation is coupledto the synthesis of ATP from ADP and phosphate. ATPsynthase is a complex structure consisting of two do-mains, F0 and F1. F1 is a spherical structure, which in thecase of mitochondria, sticks out into the matrix and isanchored to the membrane by a stator to prevent rotation.It consists of three �- and three �-subunits, all of whichcan bind nucleotides, but only the �-subunits take part inthe synthetic reactions. F0 is a cylindrical structure capableof rotation when driven by translocated protons and whichis linked to a central stalk that can revolve inside F1. Themechanism that drives ATP synthesis seems to depend ona binding charge concept in which catalytic sites on the�-subunits have different affinities for nucleotides and aredesignated loose (L), tight (T), and open (O). These arepictured in Fig. 1 on the minimap on the �-subunits col-ored blue, brown, and pink respectively. The loose (L) sitesbind the substrates (ADP and phosphate) reversibly. The Tsites then bind the reactants so tightly that ATP is formed.The O sites, which have a very low affinity for substrates,then release the ATP already formed in the T state. The

central stalk is driven by the passage of protons through F0

(counter-clockwise as seen from above), and rotates in120° stages. At each stage each of the �-subunits in turnchange conformation; L changes to T (after binding ADPand phosphate), T to O, and O to L (after releasing ATP).The new L site then binds new ADP and phosphate andbegins a new reaction sequence.

One complete revolution of F0 therefore results in theformation of 3 ATP, one from each of the �-subunits. In thisexample about 10 protons need to be translocated foreach complete revolution of F0, which means that theformation of 1 ATP requires about 3.3, though other spe-cies may be different. ATP synthase is thought to revolveat more than 100 Hz (revolutions/s), which is sufficient toproduce a turnover of the weight of our body of ATP eachday. This is what makes biochemistry meaningful, wonder-ful, and fun!

The mechanism of ATP synthase is difficult to describein words, but an animated picture has been created inPowerPoint (and real animation is proceeding), whichmakes it much easier to understand. Details of this willappear in later issues of BAMBED.

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