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 

C8. Overall Coupling Oxidation and ATP Synthesis

How is this proton gradient coupled to ATP synthesis? Another mitochondrial inner membrane complex, FoF1ATPase, also called ATP synthase or complex V,  is found in the inner mitochondrial membrane.

Figure:  FoF1ATPase, also called ATP synthase

ATPase structure

It contains two domains, a transmembrane proton channel, and a enzymatic domain which can either synthesize or hydrolyze ATP.  As protons stream through the membrane pore, conformational changes, probably mediated by concerted changes in amino acid side chain pKas. cause the protein to synthesize ATP. Based on kinetic and structural data, Boyer devised an innovative hypothesis for the mechanism of ATP synthesis, which has been verified by structural data.  In this model the enzyme, which has multiple subunits, has 3 sites for ATP binding, named L, O, and T. The L or Loose site, binds ATP loosely, the T or Tight site binds it tightly, while the O or Open site does not bind ATP.  Although the ΔGo for ATP synthesis in solution is +7.5 kcal/mol, it appears the ΔGo for bound ADP + Pi ----> bound ATP is about 1. Hence the reaction is readily reversible. The difficulty lies in dissociating the bound ATP from the complex. Initially, ADP and Pi bind to the L site. A conformational change occurs, switching the site from L to T, and concomitantly, a T site with ATP bound to an O site which promotes ATP departure. Since the T site has ADP and Pi bound, but has high affinity for ATP, it promotes the synthesis of ATP at that site. This reflects the idea that enzymes bind the transition state (which presumably looks more like ATP than ADP and Pi) more tightly than the substrate. ADP and Pi bind to the newly formed L site which promotes the switch from the T to O site, releasing ATP from the enzyme.

It should now be clear why the enzymes for oxidative phosphorylation in aerobic conditions are membrane bound. Only in this way could a proton gradient be established. Protons must be vectorially transferred in one direction only for a gradient to be established!

Figure:  Overview of metabolism: Aerobic and Anerobic Generation of NADH, Regeneration of NAD, and Coupling Oxidation/Phosphorylation

Aerobic ATP production can be uncoupled from electron transfer, as we saw with arsenate uncoupling of ox/phos in aneraobic metabolism in glycolysis.  In that case, the energy sources driving ATP synthesis was removed through hydrolysis of the mixed carboxylate/arsenate anhydride.  In aerobic metabolism, the energy source is the proton gradient.  If this gradient could be artificially collapsed, ATP synthesis would stop, but electron transport (oxidation of NADH through formation of water from dioxygen) would continue.  2,4-dinitrophenol can collapse the proton gradient and act as an uncoupler.  In the low pH milieu of the intermembrane space, this weak acid would be protonated.  It is also sufficiently nonpolar so as to have reasonable bilayer permeability.  When it reaches the higher pH matrix, it can deprotonate.  The net effect is to shunt protons through the intermembrane and not through the F0F1ATPase. 

Figure:  Uncoupling Aerobic Ox/Phos


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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